The present invention relates to monoclonal antibodies reactive to mammalian connective tissue growth factor (CTGF) or a portion thereof; cells producing the monoclonal antibodies; antibody-immobilized insoluble carriers on which the monoclonal antibodies or a portion thereof are immobilized; labeled antibodies obtained by labeling the monoclonal antibodies with labeling agents; kits for detecting, separating, assaying or purifying mammalian CTGF; methods for detecting, separating, assaying or purifying mammalian CTGF; pharmaceutical compositions comprising the monoclonal antibodies; transgenic mice to which the human CTGF gene is introduced; a polypeptide of rat CTGF; a DNA encoding rat CTGF; and antibodies reactive to rat CTGF.
Injured tissues are regenerated by the following process: removal of useless tissue fragments and cell fragments or bacteria and so on by phagocytes such as macrophages that migrate to the injured site; recovery of vessels; and the subsequent tissue renewal. Transforming growth factor xcex2(TGF-xcex2) produced by macrophages and neutrophils, which appear during the process of the tissue regeneration and recovery, has been revealed to serve as the first regulatory factor in the regeneration-recovery process.
TGF-xcex2 has multiple functions. The factor is known to regulate the production of the extracellular matrix (ECM) from connective tissue cells as well as to induce the proliferation of mesenchymal cells and to inhibit the proliferation of vascular endothelial cells and epithelial cells.
Increased production of platelet-derived growth factor (PDGF) and connective tissue growth factor (CTGF; also called Hcs24) is found in the culture supernatant of the above-mentioned mesenchymal cells of which proliferation is induced by a stimulus with TGF-xcex2. Because of this, it is presumed that the cell proliferation is not directly but indirectly induced by TGF-xcex2 with the help of other regulatory factors.
Human and mouse CTGFs have been identified previously (so far, there is no report on the identification of rat CTGF), and their physicochemical and biological properties have been analyzed ( less than human CTGF greater than : J. Cell Biology, vol. 114, No. 6, p.1285-1294, 1991; Int. J. Biochem. Cell Biol., Vol. 29, No. 1, p. 153-161, 1997; Circulation, vol. 95, No. 4, p.831-839, 1997; Cell Growth Differ., Vol. 7, No. 4, p. 469-480, 1996; J. Invest. Dermatol., Vol. 106, No. 4, p. 729-733, 1996; J. Invest. Dermatol., Vol. 105, No. 2, p. 280-284, 1995; J. Invest. Dermatol. Vol. 105, No. 1, p. 128-132, 1995; WO96/38172;  less than mouse CTGF (Fisp12) greater than : Unexamined Published Japanese Patent Application (JP-A) No. Hei 5-255397; Cell Growth Differ., vol. 2, No. 5, p. 225-233, 1991; FEBS Letters, Vol. 327, No. 2, p. 125-130, 1993; DNA Cell Biol., Vol. 10, No. 4, p. 293-308, 1991).
CTGF is a cysteine-rich secretory glycoprotein with a molecular weight of about 38 kDa. It has been revealed that the biosynthesis and secretion of the protein are induced by TGF-xcex2. CTGF has similar properties with PDGF in the light of that: their productions are induced by TGF-xcex2; they bind to the PDGF receptor and induce the proliferation of mesenchymal cells; and they are produced by fibroblasts and epithelial cells. However, they exhibit no homology at the amino acid level and thus the two molecules are distinct to each other (The Journal of Cell Biology, vol. 114, No. 6, p. 1287-1294, 1991; Molecular Biology of the Cell, Vol. 4, p.637-645, 1993).
In recent studies, low molecular weight species of CTGF have been found in the culture supernatant of human and mouse fibroblast cells as well as in the secreting fluid derived from the porcine uterus. They are biologically active but are presumed to be degradation products of 38 kDa CTGF molecules, since their molecular weights are about 10-12 kDa (Growth Factors, vol. 15, No. 3, p. 199-213, 1998; J. Biol. Chen., vol. 272, No. 32, p. 20275-20282, 1997).
Details of the relationship between physiological functions of CTGF and diseases have yet to be fully clarified. However, it has been found that: CTGF production is induced by TGF-xcex2; the expression level of CTGF mRNA is significantly high in tissues and cells derived from patients affected with various diseases (Int. J. Biochem. Cell. Biol., Vol. 29, No. 1, p. 153-161, 1997; Circulation, Vol. 95, No. 4, p. 831-839, 1997; J. Invest. Dermatol, Vol. 106, No. 4, p. 729-733, 1996; J. Invest. Dermatol., Vol. 105, No. 2, p. 128-132, 1995; J. Cell Physiol., Vol. 165, No. 3, p. 556-565, 1995; Kidney Int., Vol. 48, No. 2, p. 5001-5009, 1995); and CTGF enhances the chemotaxis and proliferation of the vascular endothelial cells (J. Cell. Biol., Vol. 114, No. 6, p. 1285-1294, 1991; Exp. Cell Res., Vol. 233, p. 63-77, 1997; Journal of Japanese Association for Oral Biology, Vol. 38, extra number, p. 463, PD0187, 1996; the 69th meeting of the Japanese Biochemical Society, proceedings, p. 683, 1P0535, 1996). These findings suggest the possibility that CTGF is associated with the onset and/or advancement of a variety of diseases.
Identification of the specific diseases awaits further findings and advancement in research. Nonetheless, CTGF has been presumed to be involved in the onset and/or advancement of a wide variety of diseases including, for example, cancers, arteriosclerosis, and skin diseases (for example, psoriasis, scleroderma, atopy, and keloid), kidney diseases, arthritis (for example, rheumatoid arthritis), various fibrotic diseases (fibrotic diseases in tissues as observed in arteriosclerosis, cirrhosis, arthritis, scleroderma, keloid, kidney fibrosis and pulmonary fibrosis, etc.).
To elucidate the association of CTGF with such various diseases, it is generally effective to detect and assay CTGF and/or the protein fragments thereof in the body fluids (serum, etc.) from patients and mammals affected with the diseases; the values determined are compared with normal values (obtained from mammals including normal persons, normal mice, normal rats and normal rabbits, etc.).
The detection and assay of secretory proteins such as CTGF are carried out by immunological assays based on antigen-antibody interaction by using the antibody (preferably used are monoclonal antibodies) which is reactive to the secretory protein to be detected; specifically, immunoassays such as radioimmunoassay (RIA) and enzyme immunoassay (EIA, ELISA) are widely used as the most convenient and useful methods for the purpose.
In this context, for the purpose of assaying CTGF, it is necessary to develop detection and assay methods using such immunoassay systems and also to prepare monoclonal antibodies against CTGF required for the establishment of assay methods. There are some reports on the preparation of antiserum reactive to CTGF (Exp. Cell Res., Vol. 233, p. 63-77, 1997; Cell Growth Differ., Vol. 8, No. 1, p. 61-68, 1997; the 69th meeting of Japanese Biochemical Society, proceedings, p. 683, 1P0534, 1996) but no report on the preparation of functional anti-CTGF monoclonal antibody which has particularly high affinity for CTGF and/or the capability of neutralizing the CTGF activity; no immunoassay systems for CTGF have so far been provided at all.
Such monoclonal antibodies having the capability of neutralizing the CTGF activity described hereinabove are useful not only as components in an immunoassay system but also as pharmaceutical antibody preparations for the treatment and/or prevention of the above-mentioned diseases caused by CTGF secretion. However, there have not been any report on such monoclonal antibodies yet.
Thus, the development of monoclonal antibodies reactive to CTGFs from various mammals such as humans, mice, rats and rabbits, has been desirably awaited. Such monoclonal antibodies are useful for the understanding of biological functions of CTGF associated with the onset and/or advancement of the above-mentioned various diseases as well as for the understanding of cause-effect relations between CTGF and the various diseases. Such monoclonal antibodies are also usable as active ingredients of pharmaceutical products for treating and preventing the diseases caused by CTGF. In particular, development of monoclonal antibodies having sufficiently high affinities for CTGF, the capability of neutralizing the CTGF activity, and/or the sufficient crossreactivity to CTGFs from a variety of mammalian species, is demanded when the antibodies are used as components in immunoassay systems for detecting CTGF to elucidate the functions of CTGF as well as the relationship between CTGF and various diseases.
In addition, it is necessary to develop monoclonal antibodies with reduced antigenicity or without antigenicity as well as with the neutralizing activity described above, when the antibodies are used for the treatment and/or prevention of various diseases in patients.
In order to fulfill the social needs, the present inventors extensively studied the monoclonal antibodies against CTGFs from a variety of mammals, and using CTGFs from various mammals as immunogens, succeeded in preparing various monoclonal antibodies against CTGFs from a variety of mammals; the antibodies are different in properties such as antigenic specificity, affinity for the antigens, the neutralizing activity and the crossreactivity.
The present inventors also succeeded, for the first time in the world, in preparing various human monoclonal antibodies against human CTGF, by immunizing, with human CTGF as an immunogen, transgenic mice created to produce human antibodies by using recombinant technology. Furthermore, the present inventors found that intact CTGFs in body fluids (serum, etc.) from a variety of mammals (human, mouse, rat, and rabbit) could be highly sensitively assayed by using various immunoassay systems constructed with the various monoclonal antibodies described above. Thus the present inventions were achieved.
The present invention was also achieved by the findings that the latter, i.e., the human antibodies, has not only the capability of significantly neutralizing the human CTGF activity, but also therapeutic effects on, for example, fibrotic diseases in tissues (kidney fibrosis, etc.) as well. The fact that these human antibodies are non-antigenic in humans, dramatically elevates the utility value of antibody as a pharmaceutical, because antigenicity is a major therapeutic problem (side effect) in medical treatment with antibody pharmaceuticals comprising antibodies derived from non-human mammals such as mice.
In particular, the present invention provides, for the first time in the field to which the present invention pertains, monoclonal antibodies that are reactive to various mammalian CTGFs and possess various useful properties as pharmaceuticals to treat and prevent diseases in patients and as components in immunoassay systems to detect and assay CTGF in body fluids from various mammals such as humans, mice, and rats.
In addition to this, the present invention provides methods and kits of immunoassay for CTGF using such various monoclonal antibodies against CTGF for the first time.
The inventive anti-human CTGF monoclonal antibodies are extremely useful as pharmaceuticals for the treatment and prevention of various diseases caused by CTGF because the antibodies are nonantigetic in humans.
By using an immunoassay with the monoclonal antibodies of the present invention, it is possible to conveniently and highly sensitively detect and assay intact CTGF in the body fluids from healthy and diseased mammals (humans, mice, rats and rabbits).
Specifically, the present inventions are defined as follows:
(1) a monoclonal antibody or a portion thereof, comprising a property in any of (a) to (g) below:
(a) reactive to human, mouse and rat connective tissue growth factors (CTGFs);
(b) reactive to both human and mouse CTGFs but not reactive to rat CTGF;
(c) reactive to both mouse and rat CTGFs but not reactive to human CTGF;
(d) inhibiting binding of human CTGF to human kidney-derived fibroblast cell line 293-T (ATCC CRL1573), or the binding of mouse CTGF to said cell line 293-T;
(e) inhibiting binding of human CTGF to any cells of rat kidney-derived fibroblast cell line NRK-49F (ATCC CRL-1570), human osteosarcoma-derived cell line MG-63 (ATCC CRL-1427), or human lung-derived fibroblasts;
(f) inhibiting cell proliferation of rat kidney-derived fibroblast cell line NRK-49F (ATCC CRL-1570)induced by a stimulus with human or mouse CTGF; or,
(g) inhibiting an increase of hydroxyproline in the kidney, wherein said hydroxyproline level tends to be elevated;
(2) the monoclonal antibody or a portion thereof according to (1), comprising a property in any of (a) to (c) below:
(a) obtainable by immunizing a mouse with human CTGF or a portion thereof, and reactive to human, mouse and rat CTGFS;
(b) obtainable by immunizing a hamster with mouse CTGF or a portion thereof, and reactive to human, mouse and rat CTGFS; or,
(c) obtainable by immunizing a rat with mouse CTGF or a portion thereof, and reactive to human, mouse and rat CTGFS;
(3) the monoclonal antibody or a portion thereof according to (1), comprising a property in any of (a) to (c) below:
(a) obtainable by immunizing a mouse with human CTGF or a portion thereof, reactive to human, mouse and rat CTGFs and inhibiting binding of human CTGF to human kidney-derived fibroblast cell line 293-T (ATCC CRL1573);
(b) obtainable by immunizing a rat with mouse CTGF or a portion thereof, reactive to human, mouse and rat CTGFs and inhibiting binding of mouse CTGF to human kidney-derived fibroblast cell line 293-T (ATCC CRL1573); or,
(c) obtainable by immunizing a hamster with mouse CTGF or a portion thereof, and reactive to human, mouse and rat CTGFs and inhibiting binding of mouse CTGF to human kidney-derived fibroblast cell line 293-T (ATCC CRL1573);
(4) the monoclonal antibody or a portion thereof according to (1), wherein said monoclonal antibody is produced by a hybridoma identified by an international deposit accession No. FERM BP-6208;
(5) the monoclonal antibody or a portion thereof according to (1), wherein said monoclonal antibody comprises a property substantially equivalent to that of a monoclonal antibody produced by a hybridoma identified by an international deposit accession No. FERM BP-6208;
(6) the monoclonal antibody or a portion thereof according to (1), wherein said monoclonal antibody is produced by a hybridoma identified by an international deposit accession No. FERM BP-6209;
(7) the monoclonal antibody or a portion thereof according to (1), wherein said monoclonal antibody comprises a property substantially equivalent to that of a monoclonal antibody produced by a hybridoma identified by an international deposit accession No. FERM BP-6209;
(8) a human monoclonal antibody or a portion thereof, reactive to any human, mouse or rat CTGF;
(9) the human monoclonal antibody or a portion thereof according to (8), wherein said human monoclonal antibody is reactive to human CTGF;
(10) a human monoclonal antibody or a portion thereof, reactive to human CTGF and comprises a property in any of (a) to (d) below:
(a) inhibiting binding of human CTGF to human kidney-derived fibroblast cell line 293-T (ATCC CRL1573);
(b) inhibiting binding of human CTGF to any of rat kidney-derived fibroblast cell line NRK-49F (ATCC CRL-1570), human osteosarcoma-derived cell line MG-63 (ATCC CRL-1427), or human lung-derived fibroblasts;
(c) inhibiting the cell proliferation of rat kidney-derived fibroblast cell line NRK-49F (ATCC CRL-1570)induced by a stimulus with human or mouse CTGF; or,
(d) inhibiting an increase of hydroxyproline in kidney, wherein said hydroxyproline level tends to be elevated;
(11) the human monoclonal antibody or a portion thereof according to any one of (8) to (10), wherein said human monoclonal antibody is derived from a non-human transgenic mammal which is capable of producing a human antibody;
(12) the human monoclonal antibody or a portion thereof according to (11), wherein said human monoclonal antibody is obtainable by immunizing a non-human transgenic mammal which is capable of producing a human antibody, with human CTGF;
(13) the human monoclonal antibody or a portion thereof according to any one of (8) to (12), wherein said non-human transgenic mammal is a transgenic mouse;
(14) the human monoclonal antibody or a portion thereof according to any one of (8) to (13), wherein a V-region DNA encoding a heavy chain variable region of said human monoclonal antibody is derived from a gene segment selected from the group consisting of DP-5, DP-38, DP-65 and DP-75;
(15) the human monoclonal antibody or a portion thereof according to any one of (8) to (13), wherein a V-region DNA encoding a light chain variable region of said human monoclonal antibody is derived from a gene segment selected from the group consisting of DPK1, DPK9, DPK12 and DPK24;
(16) the human monoclonal antibody or a portion thereof according to any one of (8) to (15), wherein a V-region DNA encoding a heavy chain variable region of said human monoclonal antibody is derived from a gene segment selected from the group consisting of DP-5, DP-38, DP-65 and DP-75, and wherein a V-region DNA encoding a light chain variable region of said human monoclonal antibody is derived from a gene segment selected from the group consisting of DPK1, DPK9, DPK12 and DPK24;
(17) the human monoclonal antibody or a portion thereof according to (9), wherein an amino acid sequence of a heavy chain variable region of said human monoclonal antibody comprises an amino acid sequence defined below in any of (a) to (j) below:
(a) the amino acid positions 21 to 120 of the amino acid sequence of SEQ ID NO: 6;
(b) the amino acid positions 21 to 120 of the amino acid sequence of SEQ ID NO: 6, wherein one or more amino acids are deleted, substituted, inserted or added;
(c) the amino acid positions 21 to 118 of the amino acid sequence of SEQ ID NO: 8;
(d) the amino acid positions 21 to 118 of the amino acid sequence of SEQ ID NO: 8, wherein one or more amino acids are deleted, substituted, inserted or added;
(e) the amino acid positions 21 to 116 of the amino acid sequence of SEQ ID NO: 10;
(f) the amino acid positions 21 to 116 of the amino acid sequence of SEQ ID NO: 10, wherein one or more amino acids are deleted, substituted, inserted or added;
(g) the amino acid positions 21 to 116 of the amino acid sequence of SEQ ID NO: 12;
(h) the amino acid positions 21 to 116 of the amino acid sequence of SEQ ID NO: 12, wherein one or more amino acids are deleted, substituted, inserted or added;
(i) the amino acid positions 21 to 117 of the amino acid sequence of SEQ ID NO: 14; or,
(j) the amino acid positions 21 to 117 of the amino acid sequence of SEQ ID NO: 14, wherein one or more amino acids are deleted, substituted, inserted or added;
(18) the human monoclonal antibody or a portion thereof according to (9), wherein an amino acid sequence of a light chain variable region of said human monoclonal antibody comprises an amino acid sequence in any of (a) to (j) below:
(a) the amino acid positions 21 to 120 of the amino acid sequence of SEQ ID NO: 16;
(b) the amino acid positions 21 to 120 of the amino acid sequence of SEQ ID NO: 16, wherein one or more amino acids are deleted, substituted, inserted or added;
(c) the amino acid positions 21 to 121 of the amino acid sequence of SEQ ID NO: 18;
(d) the amino acid positions 21 to 121 of the amino acid sequence of SEQ ID NO: 18, wherein one or more amino acids are deleted, substituted, inserted or added;
(e) the amino acid positions 23 to 117 of the amino acid sequence of SEQ ID NO: 20;
(f) the amino acid positions 23 to 117 of the amino acid sequence of SEQ ID NO: 20, wherein one or more amino acids are deleted, substituted, inserted or added;
(g) the amino acid positions 17 to 111 of the amino acid sequence of SEQ ID NO: 22;
(h) the amino acid positions 17 to 111 of the amino acid sequence of SEQ ID NO: 22, wherein one or more amino acids are deleted, substituted, inserted or added;
(i) the amino acid positions 23 to 118 of the amino acid sequence of SEQ ID NO: 24; or,
(j) the amino acid positions 23 to 118 of the amino acid sequence of SEQ ID NO: 24, wherein one or more amino acids are deleted, substituted, inserted or added;
(19) a monoclonal antibody or a portion thereof, reactive to human CTGF, which is produced by a hybridoma identified by an international deposit accession No. FERM BP-6535;
(20) a monoclonal antibody or a portion thereof, reactive to human CTGF and comprises a property substantially equivalent to that of a monoclonal antibody produced by a hybridoma identified by an international deposit accession No. FERM BP-6535;
(21)a monoclonal antibody or a portion thereof, reactive to human CTGF, and which is produced by a hybridoma identified by an international deposit accession No. FERM BP-6598;
(22) a monoclonal antibody or a portion thereof, reactive to human CTGF and comprises a property substantially equivalent to that of a monoclonal antibody produced by a hybridoma identified by an international deposit accession No. FERM BP-6598;
(23) a monoclonal antibody or a portion thereof, reactive to human CTGF, which is produced by a hybridoma identified by an international deposit accession No. FERM BP-6599;
(24) a monoclonal antibody or a portion thereof, reactive to human CTGF and comprises a property substantially equivalent to that of a monoclonal antibody produced by a hybridoma identified by an international deposit accession No. FERM BP-6599;
(25) a monoclonal antibody or a portion thereof, reactive to human CTGF, which is produced by a hybridoma identified by an international deposit accession No. FERM BP-6600;
(26) a monoclonal antibody or a portion thereof, reactive to human CTGF and comprises a property substantially equivalent to that of a monoclonal antibody produced by a hybridoma identified by an international deposit accession No. FERM BP-6600;
(27) a monoclonal antibody or a portion thereof, reactive to human CTGF, and which is non-reactive to a antigen-antibody complex of human CTGF and the monoclonal antibody reactive to human CTGF of (17) or (18);
(28) the monoclonal antibody or a portion thereof according to (27), wherein said monoclonal antibody is a human monoclonal antibody;
(29) a monoclonal antibody or a portion thereof, reactive to rat CTGF;
(30) a recombinant chimeric monoclonal antibody, reactive to human CTGF, and of which a variable region is derived from a variable region of the monoclonal antibody according to any one of (2) to (7), (27) or (29) and of which a constant region is derived from a constant region of a human immunoglobulin;
(31) a recombinant humanized monoclonal antibody, reactive to human CTGF, of which a whole or portion of the complementarity-determining regions of a hyper-variable region is derived from complementarity-determining regions of the monoclonal antibody of any one of (2) to (7), (27) or (29), of which framework regions of a hyper-variable region are derived from the framework regions of a human immunoglobulin and of which a constant region is derived from a constant region of a human immunoglobulin;
(32) a cell producing the monoclonal antibody according to any one of (1) to (29);
(33) a cell producing the recombinant monoclonal antibody according to (30) or (31);
(34) the cell according to (32), wherein said cell is a hybridoma obtainable by fusing a mammalian myeloma cell with a mammalian B cell which is capable of producing the monoclonal antibody;
(35) the cell according to (32) or (33), wherein said cell is a genetically engineered cell transformed by either one or both of the DNAs encoding a heavy chain and light chain of the monoclonal antibody;
(36) the hybridoma according to (34), wherein said hybridoma is identified by an international deposit accession No. FERM BP-6535;
(37) the hybridoma according to (34), wherein said hybridoma is identified by an international deposit accession No. FERM BP-6598;
(38) the hybridoma according to (34), wherein said hybridoma is identified by an international deposit accession No. FERM BP-6599;
(39) the hybridoma according to (34), wherein said hybridoma is identified by an international deposit accession No. FERM BP-6600;
(40) the hybridoma according to (34), wherein said hybridoma is identified by an international deposit accession No. FERM BP-6208;
(41) the hybridoma according to (34), wherein said hybridoma is identified by an international deposit accession No. FERM BP-6209;
(42) an antibody-immobilized insoluble carrier on which the monoclonal antibody according to anyone of (1) to (31) is immobilized;
(43) the antibody-immobilized insoluble carrier according to (42), wherein said insoluble carrier is selected from the group consisting of plates, test tubes, tubes, beads, balls, filters and membranes;
(44) the antibody-immobilized insoluble carrier according to (42), wherein said insoluble carrier is a filter or membrane, or that used for affinity column chromatography;
(45) a labeled antibody which is prepared by labeling the monoclonal antibody of any one of (1) to (31) with a labeling agent capable of providing a detectable signal by itself or together with other substances;
(46) the labeled antibody according to (45), wherein said labeling agent is an enzyme, fluorescent substance, chemiluminescent substance, biotin, avidin, or radioisotope;
(47) a kit for detecting or assaying mammalian CTGF, comprising at least one monoclonal antibody, an antibody-immobilized insoluble carrier, and a labeled antibody, which is selected from the group consisting of the monoclonal antibody according to any one of (1) to (31), the antibody-immobilized insoluble carrier according to (42) or (43), and the labeled antibody according to (45) or (46);
(48) the kit for detecting or assaying mammalian CTGF according to (47), comprising the antibody-immobilized insoluble carrier according to (42) or (43) and the labeled antibody according to (45) or (46);
(49) a method for detecting or assaying mammalian CTGF by an immunoassay using at least one monoclonal antibody, an antibody-immobilized insoluble carrier, and a labeled antibody, which is selected from the group consisting of the monoclonal antibody according to any one of (1) to (31), the antibody-immobilized insoluble carrier according to (42) or (43), and the labeled antibody according to (45) or (46);
(50) the method for detecting or assaying mammalian CTGF by an immunoassay according to (49), comprising at least the following steps of (a) and (b):
(a) reacting a sample with the antibody-immobilized insoluble carrier according to (42) or (43); and,
(b) reacting the labeled antibody according to (45) or (46) with an antigen-antibody complex formed by binding mammalian CTGF in said sample to the antibody-immobilized insoluble carrier;
(51) the method for detecting or assaying mammalian CTGF by an immunoassay according to (49), comprising at least the following steps of (a) and (b):
(a) reacting a sample with the labeled antibody according to (45) or (46); and,
(b) reacting the antibody-immobilized insoluble carrier according to (42) or (43) with the antigen-antibody complex formed by binding said labeled antibody and mammalian CTGF in said sample;
(52) the method for detecting or assaying mammalian CTGF by an immunoassay according to (49), comprising at least the following step of (a):
(a) reacting a mixture comprising the antibody-immobilized insoluble carrier according to (42) or (43), the labeled antibody according to (45) or (46), and a sample;
(53) the method for detecting or assaying mammalian CTGF by an immunoassay according to (49), comprising at least the following step of (a):
(a) reacting a sample and a mammalian CTGF standard labeled with a labeling agent capable of providing a detectable signal by itself or together with other substances, with the antibody-immobilized insoluble carrier according to (42) or (43);
(54) the method for detecting or assaying mammalian CTGFs by an immunoassay according to (49), comprising at least the following steps of (a) and (b):
(a) reacting the monoclonal antibody according to any one of (1) to (31) with a mixture comprising a sample and a mammalian CTGF standard labeled with a labeling agent capable of providing a detectable signal by itself or together with other substances; and,
(b) reacting a mammalian antiserum reactive to said monoclonal antibody with the antigen-antibody complex formed by binding mammalian CTGF in said sample or said labeled mammalian CTGF standard and said monoclonal antibody;
(55) the method for detecting or assaying mammalian CTGFs by an immunoassay according to (49), comprising at least the following steps of any of (a) to (c):
(a) reacting the monoclonal antibody according to any of one s (1) to (31) with a sample;
(b) reacting a mammalian CTGF standard labeled with a labeling agent capable of providing a detectable signal by itself or together with other substances with a reaction product resulted from the reaction in step (a); and,
(c) reacting a mammalian antiserum reactive to said monoclonal antibody with the antigen-antibody complex formed by binding mammalian CTGF in said sample or said labeled mammalian CTGF standard, and said monoclonal antibody;
(56) a kit for separating or purifying mammalian CTGF, comprising the antibody-immobilized insoluble carrier according to (42) or (44);
(57) a method for separating or purifying mammalian CTGF, comprising using affinity chromatography with the antibody-immobilized insoluble carrier according to (42) or (44);
(58) the purification method for mammalian CTGF according to (57), wherein said affinity chromatography is affinity column chromatography;
(59) a transgenic mouse in which DNA encoding human CTGF is integrated into an endogenous gene locus;
(60) a rat CTGF comprising an amino acid sequence of, or substantially equivalent to the amino acid sequence of SEQ ID NO: 2;
(61) a DNA encoding a rat CTGF comprising the amino acid sequence of SEQ ID NO: 2;
(62) the DNA according to (61), comprising nucleotide sequence in the positions of 213 to 1256 of SEQ ID NO: 1;
(63) a pharmaceutical composition comprising the monoclonal antibody or a portion thereof according to any one of (2) to (31) and a pharmaceutically acceptable carrier;
(64) a pharmaceutical composition comprising the human monoclonal antibody or a portion thereof according to any one of (9) to (18) or (28) and a pharmaceutically acceptable carrier;
(65) a pharmaceutical composition comprising the human monoclonal antibody or a portion thereof according to any one of (14) to (18) and (28);
(66) the pharmaceutical composition according to any one of (63) to (65), for inhibiting proliferation of cells capable of proliferating by a stimulus with CTGF;
(67) the pharmaceutical composition according to any one of (63) to (65), for treating or preventing a disease accompanied by proliferation of cells capable of proliferating by a stimulus with CTGF;
(68) the pharmaceutical composition according to (66) or (67), wherein said proliferation is cell proliferation in a tissue selected from the group consisting of brain, neck, lung, heart, liver, pancreas, kidney, stomach, large intestine, small intestine, duodenum, bone marrow, uterus, ovary, testis, prostate gland, skin, mouth, tongue and blood vessels;
(69) the pharmaceutical composition according to (68), wherein said tissue is the lung, liver, kidney or skin;
(70) the pharmaceutical composition according to (69), wherein said tissue is the kidney;
(71) the pharmaceutical composition according to (67), wherein said disease is further accompanied by tissue fibrosis;
(72) the pharmaceutical composition according to (71), wherein said tissue fibrosis is tissue fibrosis in lung, liver, kidney or skin;
(73) the pharmaceutical composition according to (72), wherein said tissue fibrosis is kidney fibrosis;
(74) a pharmaceutical composition for treating or preventing a kidney disease, comprising a CTGF inhibitor or an agent for inhibiting CTGF production, and a pharmaceutically acceptable carrier;
(75) the pharmaceutical composition according to (74), wherein said inhibitor is a monoclonal antibody reactive to CTGF;
(76) the pharmaceutical composition according to (74), wherein said inhibitor is the monoclonal antibody of any one of (9) to (31);
(77) the pharmaceutical composition according to (76), wherein said inhibitor is the human monoclonal antibody according to any one of (14) to (18) and (28);
(78) the pharmaceutical composition according to any one of (74) to (77), wherein said disease is accompanied by tissue fibrosis;
(79) a pharmaceutical composition for inhibiting proliferation of cells in kidney which are capable of proliferating by a stimulus with CTGF, comprising a substance having an activity of inhibiting proliferation of said cells and a pharmaceutically acceptable carrier;
(80) the pharmaceutical composition according to (79), wherein said substance is a monoclonal antibody reactive to CTGF;
(81) the pharmaceutical composition according to (79), wherein said inhibitor is the monoclonal antibody according to any one of (9) to (31);
(82) the pharmaceutical composition according to (81), wherein said inhibitor is the human monoclonal antibody according to any one of (14) to (18) and (28).
The present inventions are described in detail herein below by defining terminologies used herein.
Herein, xe2x80x9cmammalsxe2x80x9d mean humans, bovine, goats, rabbits, mice, rats, hamsters and guinea pigs; preferred are humans, rabbits, rats, hamsters or mice, and particularly preferred are humans, rats, hamsters or mice.
The terminologies xe2x80x9cmammals except humanxe2x80x9d and xe2x80x9cnon-human mammalsxe2x80x9d in the present invention have the same meaning, and both indicate all the above-defined mammals except humans.
xe2x80x9cAmino acidsxe2x80x9d used in the present invention mean any amino acid existing in nature and preferably the following amino acids presented by alphabetical triplets or single letter codes used to represent amino acids. (Gly/G) glycine, (Ala/A) alanine, (Val/V) valine, (Leu/L) leucine, (Ile/I) isoleucine, (Ser/S) serine, (Thr/T) threonine, (Asp/D) aspartic acid, (Glu/E) glutamic acid, (Asn/N) asparagine, (Gln/Q) glutamine, (Lys/K) lysine, (Arg/R) arginine, (Cys/C) cysteine, (Met/M) methionine, (Phe/F) phenylalanine, (Tyr/Y) tyrosine, (Trp/W) tryptophane, (His/H) histidine, (Pro/P) proline.
The term xe2x80x9cconnective tissue growth factor (CTGF)xe2x80x9d as referred to in the present invention means CTGF derived from the above-mentioned mammals, and includes, for example, human and mouse CTGFs having the above-described structure and function as reported in previous reports (for example: The Journal of Cell Biology Vol. 114, No. 6, p. 1287-1294, 1991; Molecular Biology of the Cell, Vol. 4, p. 637-645, 1993; Biochem. Biophys. Res. Comm. Vol.234, p.206-210, 1997, etc.). As a matter of course, the xe2x80x9cconnective tissue growth factorxe2x80x9d also includes rat CTGF that is included within the scope of the present invention.
Moreover, xe2x80x9cconnective tissue growth factorxe2x80x9d as referred to in the present invention includes not only the CTGF (for example, human CTGF) with a molecular weight of about 38 kDa as documented in reports but also a low-molecular-weight CTGF protein, with a molecular weight ranging from about 10 to about 12 kDa. The low-molecular-weight protein is assumed to be a degradation product of the full-length CTGF (for example, human CTGF) with a molecular weight of about 38 kDa (Growth Factors, Vol. 15, No. 3, p. 199-213, 1998; J. Biol. Chem., Vol. 272, No. 32, p. 20275-20282, 1997). Although the structure of this low-molecular-weight CTGF remains to be clarified, there is a possibility that, in the case of human CTGF, the low-molecular-weight CTGF corresponds to a C-terminal protein (molecular weight: about 11,800 Da) consisting of 103 amino acid residues resulted from the cleavage of the full-length human CTGF consisting of 349 amino acids between leucine at amino acid position 246 (Leu246) and glutamic acid at amino acid position 247 (Glu247) or another C-terminal protein (molecular weight: about 11,671 Da) consisting of 102 amino acid residues resulted from the cleavage of the full-length human CTGF between glutamic acid at amino acid position 247 (Glu247) and glutamic acid at amino acid position 248 (Glu248).
In addition, CTGF as referred to in the present invention includes CTGFs having substantially the same amino acid sequence as that of the natural CTGF (in particular, human CTGF) having the native primary structure (amino acid sequence) or a portion thereof, as long as the xe2x80x9cmonoclonal antibodyxe2x80x9d of the present invention, which is described hereinafter, is reactive to the natural CTGF or a portion thereof.
Here, xe2x80x9chaving substantially the same amino acid sequencexe2x80x9d means to include a protein having an amino acid sequence where multiple amino acids, preferably 1 to 10 amino acids, particularly preferably 1 to 5 amino acids, in the amino acid sequence of the natural CTGF protein, are substituted, deleted and/or modified, and a protein having an amino acid sequence where multiple amino acids, preferably 1 to 10 amino acids, particularly preferably 1 to 5 amino acids, are added to the amino acid sequence, as long as the protein has substantially the same biological properties as the natural CTGF protein. Furthermore, a combination of two or more of the above alterations including a substitution, deletion, modification and addition is also included.
The CTGF of the present invention can be produced by suitably using a method known in the technical field, such as recombinant technology, chemical synthesis or cell culture, or by using a modified method thereof.
The CTGF of the present invention also includes xe2x80x9ca portionxe2x80x9d of the CTGF. The terminology xe2x80x9ca portion of CTGFxe2x80x9d here refers to a polypeptide comprising any arbitrary partial amino acid sequence derived from the above-defined CTGF (including the above-mentioned low-molecular-weight CTGF of about 10 to 12 kDa). Specifically, the polypeptide includes CTGF peptide fragments with 5 to 100 amino acid residues (for example, the peptides in the C-terminus), more specifically, includes CTGF peptide fragments with 5 to 50 amino acid residues, and even more specifically the peptide fragments with 5 to 30 amino acid residues. Preferably, the polypeptide has a partial structure of CTGF comprising a domain that binds or interacts with the receptor thereof (receptor binding site, etc.) or comprising a domain necessary to the biological function of CTGF (active site, etc.).
These polypeptides (partial structures or fragments) can be produced according to a method known in the technical field, or a modified method thereof, by using recombinant technology or chemical synthesis. The polypeptides can also be produced by appropriately digesting the CTGF isolated by the cell culture method with proteases and such.
xe2x80x9cMonoclonal antibodyxe2x80x9d as referred to in the present invention is a monoclonal antibody reactive to mammalian connective tissue growth factor (CTGF) or a portion thereof. Specifically, the xe2x80x9cmonoclonal antibodyxe2x80x9d is a monoclonal antibody having a property described above in any of the inventions (1) to (31). More specifically, xe2x80x9cmonoclonal antibodyxe2x80x9d means the various monoclonal antibodies with a variety of properties and industrial utilities described below in the examples and as indicated in the drawings.
As a preferable embodiment, the monoclonal antibody of the present invention is exemplified by the following monoclonal antibodies described in (i) to (iv):
(i) the monoclonal antibody according to (1), wherein the monoclonal antibody comprises a property described in any of (d) to (g);
(ii) the monoclonal antibody according to (2);
(iii) the monoclonal antibody according to any one of (4) to (7);
(iv) the monoclonal antibody according to any one of (9) to (31).
In this embodiment, for the purpose of usage as a pharmaceutical for treating or preventing various diseases, preferable monoclonal antibody is a human monoclonal antibody included by the antibodies described above in (i) to (iv).
In this embodiment, any of the monoclonal antibodies described above in (i) to (iv) are usable for the detection, assay, separation or purification of mammalian CTGFs, which is another subject matter of the present invention.
As a more preferable embodiment, the monoclonal antibody of the present invention is exemplified by the following monoclonal antibodies described in (v) and (vi):
(v) the monoclonal antibody according to (1), wherein the monoclonal antibody comprises a property described in any of (d) to (g);
(vi) the monoclonal antibody according to any of (4) to (7), (10), and (14) to (28).
In this embodiment, for the purpose of usage as a pharmaceutical for treating or preventing various diseases, preferable monoclonal antibody is a human monoclonal antibody included in the antibodies described above in (v) and (vi).
Furthermore, in this embodiment, any of the monoclonal antibodies described above in (v) and (vi) are usable for the detection, assay, separation or purification of mammalian CTGFs which is another subject matter of the present invention.
As a particularly preferable embodiment, the monoclonal antibody of the present invention is exemplified by the following monoclonal antibodies described in (vii) and (viii):
(vii) the monoclonal antibodies described above in any of (iv) to (vii);
(viii) the monoclonal antibody according to any of (14) to (26) and (28).
In this embodiment, for the purpose of usage as a pharmaceutical for treating or preventing various diseases, preferable monoclonal antibody is the human monoclonal antibody described above in (viii).
In this embodiment, any of the monoclonal antibodies described above in (vii) and (viii)are usable for the detection, assay, separation or purification of mammalian CTGFs which is another subject matter of this invention.
As a more particularly preferable embodiment, the monoclonal antibody of the present invention is exemplified by the following monoclonal antibodies described in (ix) to (xiv):
(ix) the monoclonal antibody according to (4) or(6);
(x) the monoclonal antibody according to any of (14) to (16);
(xi) the monoclonal antibody according to (17), wherein the monoclonal antibody comprises a property described in any of (a), (c), (e), (g) and (i);
(xii) the monoclonal antibody according to (18), wherein the monoclonal antibody comprises a property described in any of (a), (c), (e), (g) and (i);
(xiii) the monoclonal antibody according to any of (19), (21) (23) and (25);
(xiv) the monoclonal antibody according to (28).
In this embodiment, for the purpose of usage as a pharmaceutical for treating or preventing various diseases, preferable monoclonal antibody is the monoclonal antibody described above in any of (x) to (xiv).
In this embodiment, any of the monoclonal antibodies described above in (ix) to (xiv) are usable for the detection, assay, separation or purification of mammalian CTGFs, which is another subject matter of the present invention. However, the monoclonal antibody described above in (ix) is particularly preferable for the purpose.
The xe2x80x9cmonoclonal antibodyxe2x80x9d of the present invention also includes a natural monoclonal antibody prepared by immunizing mammals such as mice, rats, hamsters, guinea pigs or rabbits with the above-defined connective tissue growth factor (including natural, recombinant, and chemically synthesized protein and cell culture supernatant) or a portion thereof as an antigen (immunogen); a chimeric antibody and a humanized antibody (CDR-grafted antibody) produced by recombinant technology; and a human monoclonal antibody, for example, obtained by using human antibody-producing transgenic animals.
The xe2x80x9cmonoclonal antibodyxe2x80x9d of the present invention further includes a recombinant monoclonal antibody produced by the xe2x80x9ccells producing recombinant monoclonal antibodyxe2x80x9d described hereinafter.
The monoclonal antibody includes those having any one of the isotypes of IgG, IgM, IgA (IgA1 and IgA2), IgD, or IgE. IgG (IgG1, IgG2, IgG3, and IgG4, preferably IgG2 or IgG4) or IgM is preferable. IgG is most preferred.
The polyclonal antibody (antisera) or monoclonal antibody of the present invention can be produced by known methods. Namely, mammals (including transgenic animals generated so as to produce an antibody derived from another animal species, such as the human antibody producing transgenic mice described below), preferably, mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, pigs, goats, horses, or bovine, or more preferably, mice, rats, hamsters, guinea pigs, or rabbits are immunized, for example, with an antigen mentioned above with Freund""s adjuvant, if necessary. The polyclonal antibody can be obtained from the serum obtained from the animal so immunized. The monoclonal antibodies are produced as follows. Hybridomas are produced by fusing the antibody-producing cells obtained from the animal so immunized and myeloma cells incapable of producing autoantibodies. Then the hybridomas are cloned, and clones producing the monoclonal antibodies showing the specific affinity to the antigen used for immunizing the mammal are screened.
The antibodies can also be produced using xe2x80x9crecombinant monoclonal antibody producing cellsxe2x80x9d of the present invention described below.
Specifically, the monoclonal antibody can be produced as follows. Immunizations are done by injecting or implanting once or several times the CTGF (including natural, recombinant, and synthetic proteins, and cell culture supernatant) or its fragment as mentioned above as an immunogen, if necessary, with Freund""s adjuvant, subcutaneously, intramuscularly, intravenously, through the footpad, or intraperitoneally into non-human mammals, such as mice, rats, hamsters, guinea pigs, or rabbits, preferably mice, rats or hamsters (including transgenic animals generated so as to produce antibodies derived from another animal such as the transgenic mouse producing human antibody described below). Usually, immunizations are performed once to four times every one to fourteen days after the first immunization. Antibody-producing cells are obtained from the mammal so immunized in about one to five days after the last immunization. The times and interval of the immunizations can be adequately altered according to the properties of the immunogen used.
Hybridomas that secrete a monoclonal antibody can be prepared by the method of Kxc3x6hler and Milstein (Nature, Vol.256, pp.495-497(1975)) and by its modified method. Namely, hybridomas are prepared by fusing antibody-producing cells contained in a spleen, lymph node, bone marrow, or tonsil obtained from the non-human mammal immunized as mentioned above, preferably a spleen, with myelomas without autoantibody-producing ability, which are derived from, preferably, a mammal such as mice, rats, guinea pigs, hamsters, rabbits, or humans, or more preferably, mice, rats, or humans.
For example, mouse-derived myeloma P3/X63-AG8.653 (653, ATCC No. CRL1580), P3/NSI/1-Ag4-1 (NS-1), P3/X63-Ag8.U1 (P3U1), SP2/0-Ag14 (Sp2/0, Sp2), PAI, F0, or BW5147; rat-derived myeloma 210RCY3-Ag.2.3.; or human-derived myeloma U-266AR1, GM1500-6TG-A1-2, UC729-6, CEM-AGR, D1R11, or CEM-T15 can be used as a myeloma used for the cell fusion.
Monoclonal antibody producing cells (e.g., hybridoma) can be screened by cultivating the cells, for example, in microtiter plates and by measuring the reactivity of the culture supernatant in the well in which hybridoma growth is observed, to the immunogen used for the immunization mentioned above, for example, by an enzyme immunoassay such as RIA and ELISA.
The monoclonal antibodies can be produced from hybridomas by cultivating the hybridomas in vitro or in vivo such as in the ascites of mice, rats, guinea pigs, hamsters, or rabbits, preferably mice or rats, more preferably mice and isolating the antibodies from the resulting the culture supernatant or ascites fluid of a mammal.
Furthermore, monoclonal antibodies can be obtained in a large quantity by cloning a gene encoding a monoclonal antibody from a hybridoma or xe2x80x9crecombinant monoclonal antibody producing cellsxe2x80x9d of the present invention described below, generating transgenic animals such as bovine, goats, sheep, or pigs in which the gene encoding the monoclonal antibody is integrated in its endogenous gene using transgenic animal generating technique, and recovering the monoclonal antibody derived from the antibody gene from milk of the transgenic animals (Nikkei Science, No.4, pp.78-84 (1997)).
Cultivating the cells in vitro can be performed depending on the property of cells to be cultured, on the object of a test study, and on various culture, by using known nutrient media or any nutrient media derived from known basal media for growing, maintaining, and storing the hybridomas to produce monoclonal antibodies in the culture supernatant.
Examples of basal media are low calcium concentration media such as Ham""F12 medium, MCDB153 medium, or low calcium concentration MEM medium, and high calcium concentration media such as MCDB104 medium, MEM medium, D-MEM medium, RPMI1640 medium, ASF104 medium, or RD medium. The basal media can contain, for example, sera, hormones, cytokines, and/or various inorganic or organic substances depending on the objective.
Monoclonal antibodies can be isolated and purified from the culture supernatant or ascites mentioned above by saturated ammonium sulfate precipitation, euglobulin precipitation method, caproic acid method, caprylic acid method, ion exchange chromatography (DEAE or DE52), affinity chromatography using anti-immunoglobulin column or protein A column.
The monoclonal antibody of the present invention also includes a monoclonal antibody comprising the heavy chain and/or the light chain in which either or both of the chains have deletions, substitutions or additions of one or several amino acids in the sequences thereof; xe2x80x9cseveral amino acidsxe2x80x9d as referred to here means multiple amino acid residues, specifically means one to ten amino acid residues, preferably one to five amino acid residues.
The partial modification of amino acid sequence (deletion, substitution, insertion, and addition) described above, can be introduced into the monoclonal antibody of the present invention by partially modifying the nucleotide sequence encoding the amino acid sequence. The partial modification of the nucleotide sequence can be performed by the usual method of site-specific mutagenesis (Proc. Natl. Acad. Sci. USA, Vol. 81, p. 5662-5666, 1984).
xe2x80x9cHuman monoclonal antibodyxe2x80x9d as referred to in this invention is a human monoclonal antibody reactive to the above-defined mammalian CTGFs (preferably human CTGF). The human monoclonal antibody is exemplified by the various human monoclonal antibodies with a variety of properties described below in the examples and as indicated in the drawings.
Specifically, the monoclonal antibody is a human immunoglobulin which is encoded by the human immunoglobulin gene segments in the entire region thereof including the variable region of the heavy chain (H chain), the constant region of the H chain, the variable region of the light chain (L chain) and the constant region of the L chain. The L chain is exemplified by a human xcexa chain and a human xcex chain.
The human monoclonal antibody of the present invention can be produced, for example, by immunizing, with the above-defined mammalian CTGFs, xe2x80x9cnon-human transgenic mammals which are capable of producing human antibodiesxe2x80x9d such as xe2x80x9ctransgenic mice which are capable of producing human antibodiesxe2x80x9d which can be produced by previously reported methods. By using the above-mentioned usual methods, it is possible to immunize non-human mammals, to prepare and screen hybridomas producing the antibodies, and to prepare the human monoclonal antibody in large quantities (Nature Genetics, Vol. 7, p. 13-21, 1994; Nature Genetics, Vol. 15, p. 146-156, 1997; Published Japanese Translation of PCT International Publication No. Hei 4-504365; Published Japanese Translation of PCT International Publication No. Hei7-509137; Nikkei Science, June edition, p.40-50, 1995; WO94/25585; Nature, Vol. 368, p. 856-859, 1994; Published Japanese Translation of PCT International Publication No. Hei 6-500233, etc.).
The human antibody-producing transgenic mice can be produced, specifically, for example, via the following processes; other human antibody-producing non-human transgenic mammals can be produced in the same manner.
(1) A process for preparing knockout mice in which endogenous immunoglobulin heavy chain gene has been functionally inactivated and the inactivation is done by substituting at least a portion of the endogenous gene locus of the mouse immunoglobulin heavy chain for a drug-resistance gene (the neomycin resistance gene, etc.) through homologous recombination;
(2) A process for preparing knockout mice in which endogenous gene of immunoglobulin light chain (a xcexa chain gene in particular) has been functionally inactivated and the inactivation is done by substituting at least a portion of the endogenous gene locus of the mouse immunoglobulin light chain for a drug-resistance gene (the neomycin resistance gene, etc.) through homologous recombination;
(3) A process for preparing transgenic mice in which a desired portion of the human immunoglobulin heavy chain gene locus has been integrated into a mouse chromosome, by using a vector, such as yeast artificial chromosome (YAC) vector, capable of transporting mega base genes;
(4) A process for preparing transgenic mice in which a desired portion of the human immunoglobulin light chain (a xcexa gene in particular) gene locus has been integrated into a mouse chromosome, by using a vector, such as YAC vector, capable of transporting mega base genes;
(5) A process for preparing transgenic mice in which both the mouse endogenous heavy chain and light chain gene loci have been functionally inactivated and both desired portions of the human immunoglobulin heavy chain and light chain genes loci have been integrated in a chromosome, of which preparation is achieved by crossbreeding, in arbitrary order, the knockout mice and the transgenic mice described above in (1) to (4).
The knockout mice mentioned above can be prepared by substituting any suitable region in the mouse endogenous immunoglobulin gene locus for a foreign marker gene (neomycin resistance gene, etc.) through homologous recombination so that the immunoglobulin gene locus can be inactivated so as not to cause a rearrangement of the gene locus.
For example, the method designated as positive-negative selection (PNS) can be used for the inactivation with homologous recombination (Nikkei Science, May edition, p. 52-62, 1994).
The functional inactivation of the immunoglobulin heavy chain locus can be achieved, for example, by introducing a lesion into a portion of the J region or a portion of the C region (the Cxcexc region, for example). The functional inactivation of the immunoglobulin light chain locus can also be achieved, for example, by introducing a lesion into a portion of the J region, a portion of the C region, or a region extending from the J region to the C region.
The transgenic mouse can be prepared according to the method as usually used for producing a transgenic animal (for example, see xe2x80x9cNewest Manual of Animal Cell Experimentxe2x80x9d, LIC press, Chapter 7, pp.361-408, (1990)). Specifically, for example, a transgenic mouse can be produced as follows. Hypoxanthine-guanine phosphoribosyl transferase (HPRT)-negative embryonic stem cells (ES cells) obtained from a normal mouse blastocyst is fused with a yeast cell containing an YAC vector, in which the gene encoding human immunoglobulin heavy chain locus or light chain locus, or its fragment and a HPRT gene have been inserted, by spheroplast fusion method. ES cells in which the foreign gene has been integrated into the mouse endogenous gene are screened by the HAT selection method. Then, the ES cells screened are microinjected into a fertilized egg (blastocyst) obtained from another normal mouse (Proc. Natl. Acad. Sci. USA, Vol.77, No.12, pp.7380-7384 (1980); U.S. Pat. No. 4,873,191). The blastocyst is transplanted into the uterus of another normal mouse as the foster mother. Then, chimeric transgenic mice are born from the foster mother mouse. By mating the chimeric transgenic mice with normal mice, heterozygous transgenic mice are obtained. By mating the heterozygous transgenic mice with each other, homozygous transgenic mice are obtained according to Mendel""s laws.
The xe2x80x9cchimeric monoclonal antibodyxe2x80x9d of the present invention is a monoclonal antibody prepared by genetic engineering, whose variable region is non-human mammal (e.g. mice, rats, hamsters, and so forth) immunoglobulin-derived variable region and whose constant region is human immunoglobulin-derived constant region and is exemplified by mouse/human chimeric antibody.
The constant region derived from human immunoglobulin has the amino acid sequence inherent in each isotype such as IgG (IgG1, IgG2, IgG3 and IgG4), IgM, IgA, IgD, and IgE. The constant region of the recombinant chimeric monoclonal antibody of the present invention can be that of human immunoglobulin belonging to any isotype. Preferably, it is the constant region of human IgG.
The chimeric monoclonal antibody of the present invention can be produced, for example, as follows. Needless to say, the production method is not limited thereto.
For example, mouse/human chimeric monoclonal antibody can be prepared, by referring to Experimental Medicine: SUPPLEMENT, Vol. 1.6, No.10 (1988); and Examined Published Japanese Patent Application (JP-B) No. Hei 3-73280. Namely, it can be prepared by ligating CH gene (C gene encoding the constant region of H chain) obtained from the DNA encoding human immunoglobulin to the downstream of active VH genes (rearranged VDJ gene encoding the variable region of H chain) obtained from the DNA encoding mouse monoclonal antibody isolated from the hybridoma producing the mouse monoclonal antibody, and by ligating the CL gene (C gene encoding the constant region of L chain) obtained from the DNA encoding human immunoglobulin to the downstream of active VL genes (rearranged VJ gene encoding the variable region of L chain) obtained from the DNA encoding the mouse monoclonal antibody isolated from the hybridoma, and operably inserting those into the same or different vectors in an expressible manner, followed by transformation of host cells with the expression vector, and cultivation of the transformants.
Specifically, DNAs are first extracted from mouse monoclonal antibody-producing hybridoma by the usual method, digested with appropriate restriction enzymes (for example, EcoRI and HindIII), electrophoresed (using, for example, 0.7% agarose gel), and analyzed by Southern blotting. After the electrophoresed gel is stained, for example, with ethidium bromide and photographed, the gel is given marker positions, washed twice with water, and soaked in 0.25M HCl for 15 minutes. Then, the gel is soaked in 0.4 N NaOH solution for 10 minutes with gentle stirring. The DNAs are transferred to a filter for 4 hours following the usual method. The filter is recovered and washed twice with 2xc3x97SSC. After the filter is sufficiently dried, it is baked at 75xc2x0 C. for 3 hours, treated with 0.1xc3x97SSC/0.1% SDS at 65xc2x0 C. for 30 minutes, and then soaked in 3xc3x97SSC/0.1% SDS. The filter obtained is treated with prehybridization solution in a plastic bag at 65xc2x0 C. for 3 to 4 hours.
Next, 32P-labeled probe DNA and hybridization solution are added to the bag and reacted at 65xc2x0 C. about 12 hours. After hybridization, the filter is washed under an appropriate salt concentration, reaction temperature, and time (for example, 2xc3x97SSC-0.1% SDS, room temperature, 10 minutes). The filter is put into a plastic bag with a little 2xc3x97SSC, and subjected to autoradiography after the bag is sealed. Rearranged VDJ gene and VJ gene encoding H chain and L chain of mouse monoclonal antibody respectively are identified by Southern blotting mentioned above. The region comprising the identified DNA fragment is fractionated by sucrose density gradient centrifugation and inserted into a phage vector (for example, Charon 4A, Charon 28, xcexEMBL3, xcexEMBL4, etc.). E. coli (for example, LE392, NM539, etc.) are transformed with the phage vectorto generate a genomic library. The genomic library is screened by plaque hybridization such as the Benton-Davis method (Science, Vol.196, pp.180-182 (1977)) using appropriate probes (H chain J gene, L chain (xcexa) J gene, etc.) to obtain positive clones comprising rearranged VDJ gene or VJ gene respectively. By making the restriction map and determining the nucleotide sequence of the clones obtained, it is confirmed that genes comprising the desired, rearranged VH (VDJ) gene or VL (VJ) gene have been obtained. Separately, human CH gene and human CL gene used for chimerization are isolated. For example, when a chimeric antibody with human IgG1 is produced, Cxcex31, gene is isolated as a CH gene, and Cxcexa gene is also isolated as a CL gene, are isolated. These genes can be isolated from human genomic library with mouse Cxcex31 gene and mouse Cxcexa gene, corresponding to human Cxcex31 gene and human Cxcexa gene, respectively, as probes, taking advantage of the high homology between the nucleotide sequences of mouse immunoglobulin gene and that of human immunoglobulin gene.
Specifically, DNA fragments comprising human Cxcexa gene and an enhancer region are isolated from human xcex Charon 4A HaeIII-AluI genomic library (Cell, Vol.15, pp.1157-1174 (1978)), for example, using a 3 kb HindIII-BamHI fragment from clone Ig146 (Proc. Natl. Acad. Sci. USA, Vol.75, pp.4709-4713 (1978)) and a 6.8 kb EcoRI fragment from clone MEP10 (Proc. Natl. Acad. Sci. USA, Vol.78, pp.474-478 (1981)) as probes. In addition, for example, after human fetal hepatocyte DNA is digested with HindIII and fractioned by agarose gel electrophoresis, a 5.9 kb fragment is inserted into xcex788 and then human Cxcex31 gene is isolated with the probes mentioned above.
Using mouse VH gene, mouse VL gene, human CH gene, and human CL gene so obtained, and taking promoter region and enhancer region into consideration, human CH gene is inserted downstream of mouse VH gene and human CL gene is inserted downstream of mouse VL gene in an expression vector such as pSV2gpt or pSV2neo with appropriate restriction enzymes and DNA ligase following the usual method. In this case, chimeric genes of mouse VH gene/human CH gene and mouse VL gene/human CL gene can be respectively inserted into a same or different expression vector.
Chimeric gene-inserted expression vector(s) thus prepared are introduced into myelomas (e.g., P3xc3x9763.Ag8.653 cells or SP210 cells) that do not produce antibodies by the protoplast fusion method, DEAE-dextran method, calcium phosphate method, or electroporation method. The transformants are screened by cultivating in a medium containing a drug corresponding to the drug resistance gene inserted into the expression vector and, then, cells producing desired chimeric monoclonal antibodies are obtained.
Desired chimeric monoclonal antibodies are obtained from the culture supernatant of antibody-producing cells thus screened.
The xe2x80x9chumanized monolonal antibody (CDR-grafted antibody)xe2x80x9d of the present invention is a monoclonal antibody prepared by genetic engineering and specifically means a humanized monoclonal antibody wherein a portion or the whole of the complementarity determining regions of the hyper-variable region are derived from the those of the hyper-variable region from non-human mammal (mouse, rat, hamster, etc.) monoclonal antibody, the framework regions of the variable region are derived from those of the variable region from human immunoglobulin, and the constant region is derived from that from human-immunoglobulin.
The complementarity determining regions of the hyper-variable region exists in the hyper-variable region in the variable region of an antibody and means three regions which directly binds, in a complementary manner, to an antigen (complementarity-determining residues, CDR1, CDR2, and CDR3). The framework regions of the variable region mean four comparatively conserved regions intervening upstream, downstream or between the three complementarity-determiningregions (frame work region, FR1, FR2, FR3, and FR4).
In other words, a humanized monoclonal antibody means that in which the whole region except a portion, or the whole region, of the complementarity determining regions of the hyper-variable region of a nonhuman mammal-derived monoclonal antibody have been replaced with their corresponding regions derived from human immunoglobulin.
The constant region derived from human immunoglobulin has the amino acid sequence inherent in each isotype such as IgG (IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD, and IgE. The constant region of a humanized monoclonal antibody in the present invention can be that from human immunoglobulin belonging to any isotype. Preferably, it is the constant region of human IgG. The framework regions of the constant region derived from human immunoglobulin are not particularly limited.
The humanized monoclonal antibody of the present invention can be produced, for example, as follows. Needless to say, the production method is not limited thereto.
For example, a recombinant humanized monoclonal antibody derived from mouse monoclonal antibody can be prepared by genetic engineering, referring to Published Japanese Translations of PCT International Publication No. Hei 4-506458 and Unexamined Published Japanese Patent Application (JP-A) No. Sho 62-296890. Namely, at least one mouse H chain CDR gene and at least one mouse L chain CDR gene corresponding to the mouse H chain CDR gene are isolated from hybridomas producing mouse monoclonal antibody, and human H chain gene encoding the whole region except human H chain CDR corresponding to mouse H chain CDR mentioned above and human L chain gene encoding the whole region except human L chain CDR corresponding to mouse L chain CDR mentioned above are isolated from human immunoglobulin genes.
The mouse H chain CDR gene(s) and the human H chain gene(s) so isolated are inserted, in an expressible manner, into an appropriate vector so that they can be expressed. Similarly, the mouse L chain CDR gene(s) and the human L chain gene(s) are inserted, in an expressible manner, into another appropriate vector so that they can be expressed. Alternatively, the mouse H chain CDR gene(s)/human H chain gene(s) and mouse L chain CDR gene(s)/human L chain gene(s) can be inserted, in an expressible manner, into the same expression vector so that they can be expressed. Host cells are transformed with the expression vector thus prepared to obtain transformants producing humanized monoclonal antibody. By cultivating the transformants, desired humanized monoclonal antibody is obtained from culture supernatant.
The xe2x80x9cmonoclonal antibodyxe2x80x9d of the invention includes xe2x80x9ca portionxe2x80x9d of the monoclonal antibody as well. The xe2x80x9cportion of an antibodyxe2x80x9d used in the present invention means a partial region of the antibody, preferably monoclonal antibody of the present invention as mentioned above, and specifically, means F(abxe2x80x2)2, Fabxe2x80x2, Fab, Fv (variable fragment of antibody), sFv, dsFv (disulfide stabilized Fv), or dAb (single domain antibody) (Exp. opin. Ther. Patents, Vol.6, No.5, pp.441-456 (1996)).
xe2x80x9cF(abxe2x80x2)2xe2x80x9d and xe2x80x9cFabxe2x80x2xe2x80x9d can be produced by treating immunoglobulin (monoclonal antibody) with a protease such as pepsin and papain, and means an antibody fragment generated by digesting immunoglobulin near the disulfide bonds existing between the hinge regions in each of the two H chains. For example, papain cleaves IgG upstream of the disulfide bonds existing between the hinge regions in each of the two H chains to generate two homologous antibody fragments in which an L chain composed of VL (L chain variable region) and CL (L chain constant region), and an H chain fragment composed of VH (H chain variable region) and CHxcex31 (xcex31 region in the constant region of H chain) are connected at their C terminal regions through a disulfide bond. Each of these two homologous antibody fragments is called Fabxe2x80x2. Pepsin also cleaves IgG downstream of the disulfide bonds existing between the hinge regions in each of the two H chains to generate an antibody fragment slightly larger than the fragment in which the two above-mentioned Fabxe2x80x2 are connected at the hinge region. This antibody fragment is called F(abxe2x80x2)2.
The xe2x80x9cmonoclonal antibody producing cellsxe2x80x9d or xe2x80x9crecombinant monoclonal antibody producing cellsxe2x80x9d of this invention mean any cells producing the above-described monoclonal antibody of this invention. Specific examples include the cells described in (1) to (3) below.
(1) monoclonal antibody-producing B cells that are obtainable from the above-described non-human mammal or human antibody producing transgenic mouse (or other transgenic non-human mammals) that produces a monoclonal antibody reactive with CTGF, which animal can be produced by immunizing the animal with the above-defined mammalian CTGF (preferably human CTGF) or a portion thereof or cells secreting the CTGF, etc.;
(2) the above-described hybridomas prepared by fusing antibody producing B cells obtained described above with myelomas derived from mammals; and
(3) monoclonal antibody producing transformants (recombinant cells) obtained by transforming other cells than the monoclonal antibody producing B cells and hybridomas (e.g. Chinese hamster ovarian (CHO) cells, Baby hamster kidney (BHK) cells, etc.) with genes (either the heavy chain-encoding gene or the light chain encoding gene, or both) encoding the monoclonal antibody isolated from the monoclonal antibody producing B cells or hybridomas.
The monoclonal antibody producing transformants (recombinant cells) of (3) mean recombinant cells producing a recombinant product of the monoclonal antibody produced by B cells of (1) or hybridomas of (2). These antibody producing transformants can be produced using known recombinant technology as used for the above-described chimeric monoclonal antibody and humanized monoclonal antibody.
The term xe2x80x9cmonoclonal antibody comprising a property substantially equivalent toxe2x80x9d as referred to in the present invention indicates that, when biological properties of two monoclonal antibodies are compared with each other, one monoclonal antibody is not significantly different from the other in at least the following biological properties:
(1) the reactivity to CTGF derived from a particular animal, which is used as an immunogen for immunizing a non-human mammal to prepare the monoclonal antibody;
(2) the reactivity to any CTGF derived from animals other than the particular animal (namely, crossreactivity);
(3) the properties measured by a variety of experiments described below in the examples.
The term xe2x80x9cmammalian antiserumxe2x80x9d as referred to in the present invention indicates a serum containing antibody reactive to the monoclonal antibody of the present invention or a portion thereof. The antiserum can be produced, according to the above-described method described in the production of monoclonal antibody, by immunizing mammals such as mice, rats, guineapigs, rabbits, goats, pigs orbovine, preferably rats, guinea pigs, rabbits or goats, with the above-mentioned monoclonal antibody or a portion thereof as an immunogen.
The term xe2x80x9cinsoluble carrierxe2x80x9d as referred to in the present invention indicates a supporting material thereon used for immobilizing the monoclonal antibbdy or a portion thereof (antibody fragment) of the present invention, or CTGF in samples (for example, body fluids such as plasma, culture supernatant, supernatant fluids obtained by centrifugation, etc.) by physical adsorption or chemical bonding.
The insoluble carrier is exemplified below in (A) and (B):
(A) plates, containers having internal spaces such as test tubes or tubes, beads (microbeads in particular), balls, filters or membranes, made of water-insoluble materials, for example, glass or plastics such as polystyrene resin, polycarbonate resin, silicone resin or nylon resin;
(B) insoluble carriers, used for affinity chromatography, such as cellulose carriers, agarose carriers, polyacrylamide carriers, dextran carriers, polystyrene carriers, polyvinyl alcohol carriers, poly(amino acid) carriers or porous silica carriers.
The term xe2x80x9cantibody-immobilized insoluble carrierxe2x80x9d as referred to in the present invention indicates the above-defined insoluble carrier on which the monoclonal antibody (or a portion of the antibody, namely an antibody fragment) of this invention is immobilized by physical adsorption or chemical bonding. These insoluble carriers with immobilized antibodies are usable for the detection, assay, separation or purification of CTGF in samples (for example, body fluids such as serum and plasma; culture supernatant; the supernatant fluids obtained by centrifugation, etc.).
The insoluble carriers shown above in (A) can be used for the detection and the assay; from the standpoint of the simplicity of operation and the simultaneous processing of many samples, in particular, the multi-well microtiter plates, which are made of plastics and have many wells, such as 96-well microtiter plates or 48-well microtiter plates, are used preferably as the insoluble carrier in the assay for assaying CTGF. The filters or membranes shown above in (A), or the insoluble carriers shown above in (B), are usable for the separation or the purification.
A xe2x80x9clabeling agent capable of providing a detectable signal through the reaction with the labeling agent alone or together with other substancesxe2x80x9d as referred to in this invention means a substance used for converting the monoclonal antibody or a portion thereof (antibody fragment) described above, or a CTGF standard into detectable forms; the conversion can be performed by the physical binding or chemical bonding between the labeling agent and the monoclonal antibody or a portion thereof, or between the labeling agent and the CTGF standard material.
Specifically, the labeling agent includes enzymes, fluorescent materials, chemiluminescent materials, biotin, avidin or radioisotopes, etc., more specifically, enzymes such as peroxidase (for example, horseradish peroxidase), alkaline phosphatase, xcex2-D-galactosidase, glucose oxidase, glucose-6-phosphate dehydrogenase, alcohol dehydrogenase, malate dehydrogenase, penicillinase, catalase, apo-glucose oxidase, urease, luciferase or acetylcholinesterase; fluorescent materials such as fluorescein isothiocyanate, phycobiliprotein, chelating compounds of rare-earth metals, dansyl chloride or tetramethylrhodamine isothiocyanate; radioisotopes such as 3H, 14C, 125I or 131I; biotin; avidin; or chemiluminescent materials.
Radioisotopes and fluorescent materials, even when used alone, give a detectable signal. On the other hand, enzymes, chemiluminescent materials, biotin, and avidin give no detectable signals, when used alone. In these cases, one or more substances are needed with the substances in order to give a detectable signal. For example, when the substance is an enzyme, at least a substrate for the enzyme is necessary to give a detectable signal. Various types of substrates are selectable depending on the methods for measuring the enzyme activity (colorimetry, immunofluorescence method, bioluminescence method or chemiluminescence method, etc.). For example, hydrogen peroxide is used as a substrate for peroxidase. When biotin is selected, avidin or enzyme-conjugated avidin is used for the reaction with biotin generally but not always. According to needs, various coloring agents are further used for the reaction depending on the type of the substrate.
The terminologies, xe2x80x9clabeled antibodyxe2x80x9d and xe2x80x9clabeled mammalian CTGF standardxe2x80x9d as referred to in the present invention indicate, respectively, monoclonal antibody (or antibody fragment) and CTGF labeled with the above-mentioned various labeling agents. The labeled antibody and labeled standard can be used to detect, assay, separate or purify CTGFs in samples (for example, body fluid samples such as serum and plasma; culture supernatants; or the supernatant fluids obtained by centrifugation, etc.). In the present invention, any of the above-mentioned labeling agents are usable. However, biotin or enzymes such as peroxidase are used favorably for the labeling from the standpoint of the high detection sensitivity or high assay sensitivity and the simplicity of operation.
Being different from a CTGF of an unknown concentration (amount) in a sample, the xe2x80x9cCTGF standardxe2x80x9d is a CTGF isolated previously and the standard adjustable to any desired concentration thereof to suit the purpose of each assay. For example, the standard substance can be used for the preparation of calibration curves.
The term xe2x80x9cimmunoassayxe2x80x9d as referred to in the present invention means the method of detecting or assaying the antigens in samples (for example, body fluid samples such as plasma; culture supernatants; or the supernatant fluids obtained by centrifugation) based on the principle of antigen-antibody reaction. In the present invention, for the immunoassay, one or more monoclonal antibodies (or antibody fragment(s)) to be used as the antibody in the antigen-antibody reaction are selected from the above-mentioned monoclonal antibodies (or antibody fragment) reactive to the mammalian CTGF of the present invention, the above-mentioned antibody-immobilized insoluble carrier (or antibody fragment-immobilized insoluble carrier) and the above-mentioned labeled antibody (or labeled antibody fragment) as well as the antigen is the mammalian CTGF, but otherwise previously known immunoassay methods are applicable in the assay.
Specifically, the immunoassay is exemplified by single antibody solid phase method, two-antibodies liquid phase method, two-antibodies solid phase method, sandwich method, enzyme multiplied immunoassay technique (EMIT method), enzyme channeling immunoassay, enzyme modulator mediated enzyme immunoassay (EMMIA), enzyme inhibitor immunoassay, immuno enzymometric assay, enzyme enhanced immunoassay or proximal linkage immunoassay all of which are described in xe2x80x9cEnzyme Immunoassay (3rd Ed., eds., Eiji Ishikawa et al., Igakushoin, 1987); or the one-pot method which is described in JP-B Hei 2-39747.
In this invention, any of these immunoassays can be selected appropriately to suit each assay purpose. However, from the standpoint of the simplicity of operation and/or the economical advantage, and especially when considering the clinical applicability, the sandwich method, the one pot method, the single antibody solid phase method and the two-antibodies solid phase method are preferably used in this invention; more preferable are the sandwich method and the one pot method. Particularly preferable is the sandwich method using a labeled antibody prepared by labeling the monoclonal antibody of the present invention with an enzyme or biotin as well as using an antibody-immobilized insoluble carrier prepared by immobilizing the monoclonal antibody on a multi-well microplate having many wells thereon, such as a 96-well microplate; another particularly preferable method is the one-pot method using a labeled antibody prepared by labeling the monoclonal antibody of the present invention with an enzyme or biotin as well as using an antibody-immobilized insoluble carrier prepared by immobilizing the monoclonal antibody on beads, such as microbeads, or small balls.
A specific example of a particularly preferable embodiment is the sandwich method or the one-pot method using a labeled antibody prepared by labeling the monoclonal antibody xe2x80x9c8-86-2,xe2x80x9d as indicated in FIG. 1, with an enzyme or biotin, as well as using an antibody-immobilized insoluble carrier prepared by immobilizing the monoclonal antibody xe2x80x9c8-64-6xe2x80x9d or xe2x80x9c13-51-2,xe2x80x9d as indicated in FIG. 1, on the microplate or the microbeads.
Human and mouse CTGFs can be detected or quantified in high sensitivity by immunoassays using the monoclonal antibody xe2x80x9c8-64-6xe2x80x9d-immobilized insoluble carrier, in combination with the monoclonal antibody xe2x80x9c8-86-2xe2x80x9d labeled with an enzyme or biotin. Rat CTGF (first disclosed in the present application) and mouse CTGF can be detected or assayed in high sensitivity by the immunoassay using the monoclonal antibody xe2x80x9c13-51-2xe2x80x9d-immobilized insoluble carrier, in combination with the monoclonal antibody xe2x80x9c8-86-2xe2x80x9d labeled with an enzyme or biotin.
The sandwich method, the one-pot method, the single antibody solid phase method, and the two-antibodies liquid phase method are described in detail herein below.
The sandwich method corresponds to the method described above in (50) of the present invention, and specifically, is an immunoassay that comprises at least the following steps (a) and (b):
(a) reacting a sample with the antibody-immobilized insoluble carrier of the present invention; and
(b) reacting a labeled antibody of the present invention with the antigen-antibody complex formed by the binding between the antibody-immobilized insoluble carrier and mammalian CTGF in the sample.
According to the present invention, a specific example of the method of assaying human or mouse CTGF is indicated below, in which the xe2x80x9cantibody-immobilized insoluble carrierxe2x80x9d is an xe2x80x9cantibody-immobilized microplatexe2x80x9d prepared by immobilizing the monoclonal antibody 8-64-6xe2x80x9d as indicated in FIG. 1 on a microplate, and the xe2x80x9clabeled antibodyxe2x80x9d is the monoclonal antibody xe2x80x9c8-86-2xe2x80x9d, as indicated in FIG. 1, labeled with biotin or an enzyme such as peroxidase; the method comprises, for example, the steps described below, but the method is not to be construed as being restricted thereto.
Not only mouse CTGF but also rat CTGF (first disclosed in the present application) can be assayed by the same procedures as indicated below, when the xe2x80x9cantibody-immobilized insoluble carrierxe2x80x9d is an xe2x80x9cantibody-immobilized microplatexe2x80x9d prepared by immobilizing the monoclonal antibody xe2x80x9c13-51-2xe2x80x9d as indicated in FIG. 1 on a microplate and the xe2x80x9clabeled antibodyxe2x80x9d is the monoclonal antibody xe2x80x9c8-86-2xe2x80x9d, as indicated in FIG. 1, labeled with biotin or an enzyme such as peroxidase.
(Step 1) preparing an antibody-immobilized microplate by immobilizing the monoclonal antibody xe2x80x9c18-64-6xe2x80x9d of the present invention on a microplate;
(Step 2) reacting a sample such as a human or mouse serum with the monoclonal antibody immobilized on the antibody-immobilized microplate by adding the sample to the microplate;
(Step 3) washing the microplate to remove the unreacted sample from the microplate;
(Step 4) preparing a labeled antibody by labeling the monoclonal antibody xe2x80x9c8-86-2xe2x80x9d of the present invention with biotin or an enzyme such as peroxidase;
(Step 5) reacting the labeled antibody with the antigen-antibody complex formed through the reaction between human or mouse CTGF in the sample and the monoclonal antibody immobilized on the microplate, by adding the labeled antibody to the microplate washed in Step 3;
(Step 6) washing out the unreacted labeled antibody from the microplate;
(Step 7) reacting the labeling agent moiety of the labeled antibody with a substrate selected depending on the type of the enzyme used (when the labeled antibody used in Step 5 is labeled with an enzyme such as peroxidase), avidin or enzyme-conjugated avidin avidin (when the labeled antibody used in Step 5 is labeled with biotin), by adding, if necessary together with a coloring agent, the substrate, or avidin or enzyme-conjugated avidin to the microplate;
(Step 8) reacting a substrate for the enzyme selected depending on the type of the enzyme conjugated with avidin, with the enzyme conjugated with avidin, by adding the substrate, when enzyme-conjugated avidin is used in Step 7;
(Step 9) stopping the enzyme reaction and the coloring reaction by adding a reaction stop solution into the reaction mixture of step 7 or 8; and
(Step 10) measuring the calorimetric intensity, fluorescence intensity or luminescence intensity.
The one-pot method corresponds to each of the methods described above in (50), (51) and (52) of the present invention.
Specifically, the first is the immunoassay method comprising at least the following steps (a) and (b);
(a) reacting a sample with an antibody-immobilized insoluble carrier of the present invention; and
(b) reacting a labeled antibody of the present invention with the antigen-antibody complex formed by the binding between the antibody-immobilized insoluble carrier and mammalian CTGF in the sample.
The second is the immunoassay method comprising at least the following steps (a) and (b);
(a) reacting a sample with a labeled antibody of the present invention; and
(b) reacting an antibody-immobilized insoluble carrier of the present invention with the antigen-antibody complex formed by the binding between the labeled antibody and mammalian CTGF in the sample.
The third is the immunoassay method comprising at least the following step (a);
(a) reacting a mixture of an antibody-immobilized insoluble carrier of the present invention, a labeled antibody of the present invention and a sample.
According to the present invention, a specific example of the method of assaying human or mouse CTGF is indicated below, in which the xe2x80x9cantibody-immobilized insoluble carrierxe2x80x9d is xe2x80x9cantibody-immobilized microbeadsxe2x80x9d prepared by immobilizing the monoclonal antibody xe2x80x9c8-64-6xe2x80x9d as indicated in FIG. 1 on microbeads, and the xe2x80x9clabeled antibodyxe2x80x9d is the monoclonal antibody xe2x80x9c8-86-2xe2x80x9d, as indicated in FIG. 1, labeled with biotin or an enzyme such as peroxidase; the method comprises, for example, the steps described below, but the method is not to be construed as being restricted thereto.
Not only mouse CTGF but also rat CTGF (first disclosed in the application) can be assayed by the same procedures as indicated below, when the xe2x80x9cantibody-immobilized insoluble carrierxe2x80x9d is xe2x80x9cantibody-immobilized microbeadsxe2x80x9d prepared by immobilizing the monoclonal antibody xe2x80x9c13-51-2xe2x80x9d as indicated in FIG. 1 on microbeads and the xe2x80x9clabeled antibodyxe2x80x9d is the monoclonal antibody xe2x80x9c8-86-2xe2x80x9d, as indicated in FIG. 1, labeled with biotin or an enzyme such as peroxidase.
The first method comprises the following steps:
(Step 1) preparing antibody-immobilized microbeads by immobilizing the monoclonal antibody xe2x80x9c8-64-6xe2x80x9d, of the present invention on microbeads;
(Step 2) reacting a sample such as a human or mouse serum with the monoclonal antibody immobilized on the microbeads by adding the sample and the antibody-immobilized microbeads together with a buffer solution into a container having internal spaces such as a test tube, microplate, or tube;
(Step 3) washing the beads to remove the liquid content from the container;
(Step 4) preparing a labeled antibody by labeling the monoclonal antibody xe2x80x9c8-86-2xe2x80x9d with biotin or an enzyme such as peroxidase;
(Step 5) reacting the labeled antibody with the antigen-antibody complex formed through the reaction between the human or mouse CTGF in the sample and the monoclonal antibody immobilized on the beads by adding the labeled antibody into the container containing the beads washed in Step 3;
(Step 6) removing the unreacted labeled antibody by removing the liquid content from the container and washing the beads;
(Step 7) reacting the labeling agent moiety of the labeled antibody with a substrate selected depending on the type of the enzyme used (when the labeled antibody used in Step 5 is labeled with an enzyme such as peroxidase), avidin or the enzyme-conjugated avidin (when the labeled antibody used in Step 5 is labeled with biotin) by adding, if necessary together with a coloring agent, the substrate, or avidin or enzyme-conjugated avidin into the container containing the beads washed in Step 6;
(Step 8) reacting a substrate for the enzyme selected depending on the type of the enzyme conjugated with avidin, with the enzyme conjugated with avidin by adding the substrate, when enzyme-conjugated avidin is used in Step 7;
(Step 9) stopping the enzyme reaction and the coloring reaction by adding a reaction stop solution into the reaction mixture of Step 7 or Step 8; and
(Step 10) measuring the calorimetric intensity, fluorescence intensity or luminescence intensity.
The second method comprises the following steps:
(Step 1) preparing a labeled antibody by labeling the monoclonal antibody xe2x80x9c8-86-2xe2x80x9d, of the present invention with biotin or an enzyme such as peroxidase;
(Step 2) reacting a sample such as a human or mouse serum with the labeled antibody by adding the sample and the labeled antibody together with a buffer solution into a container having internal spaces such as a test tube, microplate or tube;
(Step 3) preparing antibody-immobilized microbeads by immobilizing the monoclonal antibody xe2x80x9c8-64-6xe2x80x9d of the present invention on microbeads;
(Step 4) reacting the monoclonal antibody immobilized on the beads with the antigen-antibody complex formed through the reaction between the labeled antibody and human CTGF or mouse CTGF in the sample by adding the beads into the reaction system in Step 3;
(Step 5) removing the unreacted labeled antibody by removing the liquid content from the container and washing the beads;
(Step 6) reacting the labeling agent moiety of the labeled antibody with a substrate selected depending on the type of the enzyme used (when the labeled antibody used in Step 2 is labeled with an enzyme such as peroxidase), avidin or the enzyme-conjugated avidin (when the labeled antibody used in Step 2 is labeled with biotin) by adding, if necessary together with a coloring agent, the substrate, or avidin or enzyme-conjugated avidin into the container containing the beads washed in Step 5;
(Step 7) reacting a substrate selected depending on the type of the enzyme conjugated with avidin, with the enzyme conjugated with avidin by adding the substrate, when enzyme-conjugated avidin is used in Step 6;
(Step 8) stopping the enzyme reaction and the coloring reaction by adding a stop solution to the reaction system in Step 6 or 7; and (Step 9) measuring the colorimetric intensity, fluorescence intensity or luminescence intensity.
The third method comprises the following steps:
(Step 1) preparing antibody-immobilized microbeads by immobilizing the monoclonal antibody xe2x80x9c8-64-6xe2x80x9d, of the present invention on the microbeads;
(Step 2) preparing a labeled antibody by labeling the monoclonal antibody xe2x80x9c8-86-2xe2x80x9d of the present invention with biotin or an enzyme such as peroxidase;
(Step 3) reacting the labeled antibody and a sample such as a human or mouse serum simultaneously with the monoclonal antibody immobilized on microbeads by adding the sample and the antibody-immobilized microbeads prepared in Step 1 and the labeled antibody prepared in Step 2 together with a buffer solution into a container having internal spaces such as a test tube, plate, or tube.
(Step 4) removing the unreacted labeled antibody by removing the liquid content from the container and washing the beads;
(Step 5) reacting the labeling agent moiety of the labeled antibody with a substrate selected depending on the type of the enzyme used (when the labeled antibody used in Step 3 is labeled with an enzyme such as peroxidase), avidin or the enzyme-conjugated avidin (when the labeled antibody used in Step 3 is labeled with biotin) by adding, if necessary together with a coloring agent, the substrate, or avidin or enzyme-conjugated avidin into the container containing the beads washed in Step 4;
(Step 6) reacting a substrate selected depending on the type of the enzyme conjugated with avidin, with the enzyme conjugated with avidin by adding the substrate, when enzyme-conjugated avidin is used in Step 5;
(Step 7) stopping the enzyme reaction and the coloring reaction by adding a stop solution to the reaction system in Step 5 or 6; and
(Step 8) measuring the colorimetric intensity, fluorescence intensity or luminescence intensity.
The single antibody solid phase method corresponds to the method described above in (53) of the present invention, and specifically, the immunoassay method that comprises at least the following step (a):
(a) reacting a sample and mammalian CTGF standard labeled with a labeling agent capable of providing a detectable signal by itself or by reacting with other substances, with an antibody-immobilized insoluble carrier of the present invention.
A specific example of the method of assaying human or mouse CTGF according to the present invention is indicated below, in which the xe2x80x9cantibody-immobilized insoluble carrierxe2x80x9d is an xe2x80x9cantibody-immobilized microplatexe2x80x9d prepared by immobilizing the monoclonal antibody xe2x80x9c8-64-6xe2x80x9d as indicated in FIG. 1 on a microplate and widely used biotin or enzyme such as peroxidase is used here as a xe2x80x9clabeling agentxe2x80x9d; the method comprises, for example, the steps described below, but the method is not to be construed as being restricted thereto. Not only mouse CTGF but also rat CTGF (first disclosed in the application) can be assayed by the same procedures as indicated below, when the xe2x80x9cantibody-immobilized insoluble carrierxe2x80x9d is an xe2x80x9cantibody-immobilized microplatexe2x80x9d prepared by immobilizing the monoclonal antibody xe2x80x9c13-51-2xe2x80x9d as indicated in FIG. 1 on a microplate and widely used biotin or an enzyme such as peroxidase is use as the xe2x80x9clabeling agent.xe2x80x9d
(Step 1) preparing an antibody-immobilized microplate by immobilizing the monoclonal antibody xe2x80x9c8-64-6xe2x80x9d on a microplate;
(Step 2) preparing a labeled CTGF standard by labeling the standard with biotin or an enzyme such as peroxidase;
(Step 3) reacting a sample such as a human or mouse serum and the labeled CTGF standard competitively with the monoclonal antibody immobilized on the microplate by adding the sample and the labeled standard to the microplate;
(Step 4) washing out the unreacted labeled standard from the microplate;
(Step 5) reacting the labeling agent moiety of the labeled standard with a substrate selected from depending on the type of the enzyme used (when the labeled standard used in Step 3 is labeled with an enzyme such as peroxidase), avidin or enzyme-conjugated avidin (when the labeled standard in Step 3 is labeled with biotin) by adding, if necessary together with a coloring agent, the substrate, or avidin or enzyme-conjugated avidin to the microplate washed in Step 4;
(Step 6) reacting a substrate selected depending on the type of the enzyme conjugated with avidin, with the enzyme conjugated with avidin by adding the substrate, when enzyme-conjugated avidin is used in Step 5;
(Step 7) stopping the enzyme reaction and the coloring reaction by adding a stop solution to the microplate; and
(Step 8) measuring the colorimetric intensity, fluorescence intensity or luminescence intensity.
The two antibodies solid phase method corresponds to the methods described above in (54) and (55) of the present invention.
Specifically, the first is the immunoassay method comprising at least the following steps (a) and (b):
(a) reacting the monoclonal antibody of the present invention with a mixture comprising a sample and a mammalian CTGF standard labeled with a labeling agent capable of providing a detectable signal by itself or by reacting with other substances; and,
(b) reacting a mammalian antiserum reactive to the monoclonal antibody with the antigen-antibody complex formed through binding of the monoclonal antibody and the mammalian CTGF in the sample or the labeled mammalian CTGF standard.
The second is the immunoassay method comprising at least the following steps (a) to (c):
(a) reacting a monoclonal antibody of the present invention with a sample;
(b) reacting a mammalian CTGF standard labeled with a labeling agent capable of providing a detectable signal by itself or by reacting other substances, with the reaction mixture in Step (a); and,
(c) reacting a mammalian antiserum reactive to the monoclonal antibody with the antigen-antibody complex formed through the binding of the monoclonal antibody and the mammalian CTGF in the sample or the labeled mammalian CTGF standard.
A specific example of the method of assaying human or mouse CTGF according to the present invention is indicated below, in which the xe2x80x9cmonoclonal antibodyxe2x80x9d is the monoclonal antibody xe2x80x9c8-64-6xe2x80x9d or the monoclonal antibody xe2x80x9c8-86-2xe2x80x9d, as indicated in FIG. 1 and widely used biotin or an enzyme such as peroxidase is used as the xe2x80x9clabeling agentxe2x80x9d; the method comprises, for example, the steps described below, but the method is not to be construed as being restricted thereto.
Not only mouse CTGF but also rat CTGF (first disclosed in the application) can be assayed by the same procedures as indicated below, when the xe2x80x9cmonoclonal antibodyxe2x80x9d is the monoclonal antibody xe2x80x9c13-51-2xe2x80x9d as indicated in FIG. 1 and widely used biotin or an enzyme such as peroxidase is used as the xe2x80x9clabeling agent.xe2x80x9d
The first method comprises the following steps:
(Step 1) preparing the labeled CTGF standard by labeling a human or mouse CTGF standard with biotin or an enzyme such as peroxidase;
(Step 2) reacting a sample such as a human or mouse serum and the labeled CTGF standard prepared in Step 1 competitively with the monoclonal antibody xe2x80x9c18-64-6xe2x80x9d or xe2x80x9c8-86-2xe2x80x9d of the present invention by adding a mixture comprising the sample and the labeled CTGF standard into a container having internal spaces such as a test tube, plate or tube and by subsequently adding thereto the monoclonal antibody;
(Step 3) reacting an antiserum, derived from mammals except mice, reactive to the mouse monoclonal antibody, such as a goat anti-mouse xcex3-globulin antiserum, with the antigen-antibody complex, formed in Step 2, consisting of the monoclonal antibody and the mammalian CTGF in the sample or the labeled mammalian CTGF standard, to give the resulting precipitated immune-complex;
(Step 4) separating the precipitated complex by the centrifugation of the reaction mixture of Step 3;
(Step 5) reacting the labeling agent moiety of the labeled standard with a substrate selected depending on the type of the enzyme used (when the labeled standard used in Step 2 is labeled with an enzyme such as peroxidase), avidin or the enzyme-conjugated avidin (when the labeled standard used in Step 2 is labeled with the biotin) by adding, if necessary together with a coloring agent, the substrate, or avidin or enzyme-conjugated avidin to the precipitated complex separated in Step 4;
(Step 6) reacting a substrate selected depending on the type of the enzyme conjugated with avidin, with the enzyme conjugated with avidin by adding the substrate, when enzyme-conjugated avidin is used in Step 5;
(Step 7) stopping the enzyme reaction and the coloring reaction by adding a stop solution to the reaction system in Step 5 or 6; and,
(Step 8) measuring the colorimetric intensity, fluorescence intensity or luminescence intensity.
The second method comprises the following steps:
(Step 1) preparing a labeled CTGF standard by labeling a human or mouse CTGF standard with biotin or an enzyme such as peroxidase;
(Step 2) reacting a sample such as a human or mouse serum with the monoclonal antibody xe2x80x9c8-64-6xe2x80x9d or xe2x80x9c8-86-2xe2x80x9d of the present invention by adding the sample into a container having internal spaces such as a test tube, plate or tube and by subsequently adding thereto the monoclonal antibody;
(Step 3) reacting the labeled CTGF standard prepared in Step 1 with the remaining unreacted monoclonal antibody, by adding the labeled CTGF standard to the reaction mixture in Step 2;
(Step 4) reacting an antiserum, derived from mammals except mice, reactive to the mouse monoclonal antibody, such as a goat anti-mouse xcex3-globulin antiserum, with the antigen-antibody complex, formed in Step 2, consisting of the monoclonal antibody and the mammalian CTGF in the sample and/or the antigen-antibody complex, formed in Step 3, consisting of the monoclonal antibody and the labeled mammalian CTGF standard, to give the precipitated immune-complex consisting of the antiserum and the antigen-antibody complex;
(Step 5) separating the precipitated complex by the centrifugation of the reaction mixture of Step 4;
(Step 6) reacting the labeling agent moiety of the labeled standard with a substrate selected depending on the type of the enzyme used (when the labeled standard used in Step 3 is labeled with an enzyme such as peroxidase), avidin or the enzyme-conjugated avidin (when the labeled standard used in Step 3 is labeled with biotin) by adding, if necessary together with a coloring agent, the substrate, or avidin or enzyme-conjugated avidin to the precipitated complex separated in Step 5;
(Step 7) reacting a substrate selected depending on the type of the enzyme conjugated with avidin, with the enzyme conjugated with avidin by adding the substrate, when enzyme-conjugated avidin is used in Step 6;
(Step 8) stopping the enzyme reaction and the coloring reaction are stopped by adding a stop solution to the reaction system in Step 6 or 7, and;
(Step 9) measuring the colorimetric intensity, fluorescence intensity or luminescence intensity.
The xe2x80x9caffinity chromatographyxe2x80x9d as referred to in the present invention indicates the method of separating or purifying the materials of interest in samples (for example, the body fluid samples such as a serum and plasma; culture supernatants; or the supernatant fluids obtained by centrifugation, etc.) by utilizing the interaction (affinity) between a pair of materials, for example, antigen and antibody, enzyme and substrate, or receptor and ligand.
The method of the present invention relates to the method for separating or purifying the mammalian CTGFs in samples (for example, the body fluid samples such as a serum and plasma; culture supernatants; or the supernatant fluids obtained by centrifugation, etc.) by utilizing the antigen-antibody interaction, specifically, the affinity of the monoclonal antibody of the present invention for mammalian CTGFs as antigens; specifically includes,
(1) a method for separating CTGF in samples by immobilizing the monoclonal antibody (or antibody fragment) reactive to mammlian CTGF on the above-defined insoluble carriers, such as a filter or a membrane and contacting the sample with the filter or membrane; and
(2) a method for separating or purifying CTGF in the samples, by immobilizing, in a usual manner (immobilization by physical adsorption, cross-linking to the carrier polymer, trapping in the carrier matrix or non-covalent bonding, etc.), the inventive monoclonal antibody (or the antibody fragment) reactive to mammalian CTGF on insoluble carriers such as cellulose carriers, agarose carriers, polyacrylamide carriers, dextran carriers, and polystyrene carriers, polyvinyl alcohol carriers, poly(amino acid) carriers or porous silica carriers; by filling a column made of glass, plastics, or stainless, with the insoluble carriers; and by loading and eluting samples (for example, the body fluid samples such as a serum and plasma; culture supernatants; or the supernatant fluids obtained by centrifugation, etc.) through the column (for example, the cylindrical column). The method described above in (2) is in particular designated as affinity column chromatography.
Any of the insoluble carriers are usable as insoluble carriers for affinity column chromatography, as long as the monoclonal antibody (or antibody fragment) of the present invention can be immobilized on the carriers. Such carriers include, for example, commercially available carriers such as SEPHAROSE 2B, SEPHAROSE 4B, SEPHAROSE 6B, CNBR-ACTIVATED SEPHAROSE 4B, AH-SEPHAROSE 4B, CH-SEPHAROSE 4B, ACTIVATED CH-SEPHAROSE 4B, EPOXY-ACTIVATED SEPHAROSE 6B, ACTIVATED THIOL-SEPHAROSE 4B, SEPHADEX, CM-SEPHADEX, ECH-SEPHAROSE 4B, EAH-SEPHAROSE 4B, NHS-ACTIVATED SEPHAROSE or THIOPROPYL SEPHAROSE 6B, etc., all of which are supplied by Pharmacia; BIO-GEL A, CELLEX, CELLEX AE, CELLEX-CM, CELLEX PAB, BIO-GEL P, HYDRAZIDE BIO-GEL P, AMINOETHYL BIO-GEL P, BIO-GEL CM, AFFI-GEL 10, AFFI-GEL 15, AFFI-PREP10, AFFI-GEL HZ, AFFI-PREP HZ, AFFI-GEL 102, CM BIO-GEL A, AFFI-GEL HEPARIN, AFFI-GEL 501 OR AFFI-GEL 601, etc., all of which are supplied by Bio-Rad; CHROMAGEL A, CHROMAGEL P, ENZAFIX P-HZ, ENZAFIX P-SH OR ENZAFIX P-AB, etc., all of which are supplied by Wako Pure Chemical Industries Ltd.; AE-CELLUROSE, CM-CELLUROSE or PAB CELLUROSE etc., all of which are supplied by Serva.
The term xe2x80x9cpharmaceutical compositionxe2x80x9d as referred to in the present invention means a composition useful as a pharmaceutical comprising as an active ingredient the monoclonal antibody of the present invention or a portion thereof, or any of the after-mentioned xe2x80x9cCTGF inhibitorxe2x80x9d, xe2x80x9cCTGF production inhibitorxe2x80x9d and xe2x80x9csubstance with the activity to inhibit the CTGF-stimulated proliferation of cells having the capability of proliferating by CTGF stimulationxe2x80x9d, as well as comprising a xe2x80x9cpharmaceutically acceptable carrier.xe2x80x9d
The xe2x80x9cpharmaceutically acceptable carrierxe2x80x9d includes an excipient, a diluent, an expander, a disintegrating agent, a stabilizer, a preservative, a buffer, an emulsifier, an aromatic, a colorant, a sweetener, a viscosity increasing agent, a flavor, a dissolving agent, or other additives.
Using one or more of such carriers, a pharmaceutical composition can be formulated into tablets, pills, powders, granules, injections, solutions, capsules, troches, elixirs, suspensions, emulsions, or syrups.
The pharmaceutical composition can be administered orally or parenterally. Other forms for parenteral administration include a solution for external application, suppository for rectal administration, and pessary, prescribed by the usual method, which comprises one or more active ingredient.
The dosage can vary depending on the age, sex, weight, and symptoms of a patient, effect of treatment, administration route, period of treatment, or the kind of active ingredient (protein or antibody mentioned above) contained in the pharmaceutical composition. Usually, the pharmaceutical composition can be administered to an adult in a dose of 10 xcexcg to 1000 mg (or 10 xcexcg to 500 mg) per one administration. Depending on various conditions, the lower dosage may be sufficient in some cases, and a higher dosage may be necessary in other cases.
In particular, the injection can be produced by dissolving or suspending the antibody in a non-toxic, pharmaceutically acceptable carrier such as physiological saline or commercially available distilled water for injections by adjusting the concentration to 0.1 xcexcg antibody/ml carrier to 10 mg antibody/ml carrier.
The injection thus produced can be administered to a human patient in need of treatment in a dose of 1 xcexcg to 100 mg/kg body weight, preferably 50 xcexcg to 50 mg/kg body weight, once or more times a day. Examples of administration routes are medically appropriate administration routes such as intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, or intraperitoneal injection, preferably intravenous injection.
The injection can also be prepared into a non-aqueous diluent (for example, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and alcohols such as ethanol), suspension, or emulsion.
The injection can be sterilized by filtration with a bacteria-non-penetratable filter, by mixing bacteriocide, or by irradiation. The injection can be prepared at the time of use. Namely, it is freeze-dried to make a sterile solid composition, and can be dissolved in sterile distilled water for injection or another solvent before use.
The pharmaceutical composition of the present invention is useful for inhibiting the proliferation of various cells having the capability of proliferating (for example, various fibroblast cells, various vascular endothelial cells, and others, etc.) in response to the stimulation of CTGFs from a variety of tissues. Examples of the tissues are, the brain, neck, lung, heart, liver, pancreas, kidney, stomach, large intestine, small intestine, duodenum, bone marrow, uterus, ovary, testis, prostate, skin, mouth, tongue, and blood vessels, and preferably, the lung, liver, kidney or skin.
As described hereinabove, the pharmaceutical composition of the present invention can inhibit the proliferation of cells having the capability of proliferating in response to the stimulation of CTGF. Therefore, the pharmaceutical composition of the present invention is also useful as a pharmaceutical for treating or preventing a variety of diseases associated with the cell proliferation in various tissues mentioned above. Examples of such tissues are the brain, neck, lung, heart, liver, pancreas, kidney, stomach, large intestine, small intestine, duodenum, bone marrow, uterus, ovary, testis, prostate, skin, mouth, tongue, and blood vessels, and preferably, the lung, liver, kidney or skin.
The diseases, to which the pharmaceutical composition of the present invention is applicable for the treatment or prevention, are, for example, fibrotic diseases in various tissues (kidney fibrosis, pulmonary fibrosis, hepatic fibrosis, fibrosis in the skin, etc.), kidney diseases (for example, kidney fibrosis, nephritis, renal failure, etc.), lung diseases (for example, pulmonary fibrosis, pneumonia, etc.), skin diseases (for example, psoriasis, scleroderma, atopy, keloid, etc.), liver diseases (for example, hepatic fibrosis, hepatitis, cirrhosis, etc.), arthritis (for example, rheumatoid arthritis), various cancers, or arteriosclerosis.
Preferable examples of the diseases are kidney diseases (for example, kidney fibrosis, nephritis, renal failure, etc.), lung diseases (for example, pulmonary fibrosis, pneumonia, etc.), skin diseases (for example, psoriasis, scleroderma, atopy, keloid, etc.), liver diseases (for example, hepatic fibrosis, hepatitis, cirrhosis, etc.).
More preferable are kidney diseases (for example, kidney fibrosis, nephritis, renal failure, etc.).
The pharmaceutical composition of the present invention includes the pharmaceutical composition comprising a xe2x80x9cCTGF inhibitor,xe2x80x9d a xe2x80x9cCTGF production inhibitorxe2x80x9d or a xe2x80x9csubstance with the activity to inhibit the CTGF-stimulated proliferation of the cells having the capability of proliferating by CTGF stimulation.xe2x80x9d
Each of the xe2x80x9cCTGF inhibitor,xe2x80x9d the xe2x80x9cCTGF production inhibitor,xe2x80x9d and the xe2x80x9csubstancexe2x80x9d means a substance having the activity of suppressing or inhibiting the biological function of CTGF, or a substance having the activity of suppressing or inhibiting the production of CTGF in a variety of cells. Such substances are exemplified by a substance having any of the following activities:
(1) the activity of suppressing or inhibiting the binding of human kidney-derived fibroblast cells (for example, cell line 293-T (ATCC CRL1573)) to human CTGF, or the binding of the cells to mouse CTGF;
(2) the activity of suppressing or inhibiting the binding of human CTGF with rat kidney-derived fibroblast cells (for example, cell line NRK-49F (ATCC CRL-1570)), human osteosarcoma cell line MG-63 (ATCC CRL-1427), or human lung-derived fibroblast cells;
(3) the activity of suppressing or inhibiting the proliferation of rat kidney-derived fibroblast cells (for example, cell line NRK-49F (ATCC CRL-1570)) in response to the stimulation of human CTGF or mouse CTGF;
(4) the activity of suppressing or inhibiting an increase of hydroxyproline in the kidney where the synthesis of hydroxyproline level tends to be increased.
Specifically, the above-mentioned xe2x80x9csubstancexe2x80x9d is exemplified by the following substances:
(a) the above-mentioned monoclonal antibody of the present invention (which is not restricted to the wild-type antibody and the recombinant antibody) or a portion thereof;
(b) antisense DNA;
(c) antisense RNA;
(d) low molecular weight chemical substances (chemically synthesized compounds or naturally-occurring substances) other than the substances indicated in (a) to (c).
The antisense DNA as referred to in the present invention includes a DNA comprising a partial nucleotide sequence of a DNA encoding the mammalian (preferably human) CTGF protein or a DNA corresponding to the above DNA that is chemically modified in part, or a DNA comprising a complementary sequence to the partial nucleotide sequence, or a DNA corresponding to this DNA that is chemically modified in part.
The xe2x80x9cpartial nucleotide sequencexe2x80x9d as referred to here indicates a partial nucleotide sequence comprising an arbitrary number of nucleotides contained in an arbitrary region of the DNA sequence encoding the mammalian (preferably human) CTGF protein.
The DNA, hybridizing to a DNA or an RNA encoding the CTGF protein, can inhibit the CTGF production by suppressing transcription of the DNA to mRNA or suppressing the translation of the mRNA into the protein.
The partial nucleotide sequence consists of 5 to 100 consecutive nucleotides, preferably 5 to 70 consecutive nucleotides, more preferably 5 to 50 consecutive nucleotides, and still more preferably 5 to 30 consecutive nucleotides.
When the DNA is used as an antisense DNA pharmaceutical, the DNA sequence can be modified chemically in part for extending the half-life (stability) of the blood concentration of the DNA administered to patients, for increasing the intracytoplasmic-membrane permeability of the DNA, or for increasing the degradation resistance or the absorption of the orally administered DNA in the digestive organs. The chemical modification includes, for example, the modification of the phosphate bonds, the riboses, the nucleotide bases, the sugar moiety, the 3xe2x80x2 end and/or the 5xe2x80x2 end in the structure of the oligonucleotide DNA.
The modification of phosphate bond includes, for example, the conversion of one or more of the bonds to phosphodiester bonds (D-oligo), phosphorothioate bonds, phosphorodithioate bonds (S-oligo), methyl phosphonate (MP-oligo), phosphoroamidate bonds, non-phosphate bonds or methyl phosphonothioate bonds, or combinations thereof. The modification of the ribose includes, for example, the conversion to 2xe2x80x2-fluororibose or 2xe2x80x2-O-methylribose. The modification of the nucleotide base includes, for example, the conversion to 5-propynyluracil or 2-aminoadenine.
The antisense RNA as referred to in the present invention includes an RNA comprising a partial nucleotide sequence of an RNA encoding mammalian (preferably human) CTGF protein or an RNA corresponding to the RNA which is chemically modified in part, or an RNA comprising a complementary sequence to the partial nucleotide sequence or an RNA corresponding to this RNA which is chemically modified in part.
The xe2x80x9cpartial nucleotide sequencexe2x80x9d as referred to here indicates a partial nucleotide sequence comprising an arbitrary number of nucleotides contained in an arbitrary region of the RNA sequence encoding mammalian (preferably human) CTGF protein.
The RNA, hybridizing to a DNA or an RNA encoding the CTGF protein, can inhibit the CTGF production by inhibiting the transcription of the DNA to mRNA or inhibiting the translation of the mRNA into the protein.
The partial nucleotide sequence consists of 5 to 100 consecutive nucleotides, preferably 5 to 70 consecutive nucleotides, more preferably 5 to 50 consecutive nucleotides, and still more preferably 5 to 30 consecutive nucleotides.
When the RNA is used as an antisense RNA pharmaceutical, the RNA sequence can be modified chemically in part for extending the half-life (stability) of the blood concentration of the RNA administered to patients, for increasing the intracytoplasmic-membrane permeability of the RNA, or for increasing the degradation resistance or the absorption of the orally administered RNA in the digestive organ. The chemical modification includes, for example, the modification of the phosphate bonds, the riboses, the nucleotide bases, the sugar moiety, the 3xe2x80x2 end and/or the 5xe2x80x2 end in the structure of the oligonucleotide RNA.
The modification of phosphate bonds includes, for example, the conversion of one or more of the bonds to phosphodiester bonds (D-oligo), phosphorothioate bond, phosphorodithioate bonds (S-oligo), methyl phosphonate (MP-oligo), phosphoroamidate bonds, non-phosphate bonds or methyl phosphonothioate bonds, or combinations thereof. The modification of the ribose includes, for example, the conversion to 2xe2x80x2-fluororibose or 2xe2x80x2-O-methylribose. The modification of the nucleotide base includes, for example, the conversion to 5-propynyluracil or 2-aminoadenine.
The therapeutic effects of the pharmaceutical composition of the present invention on various diseases can be examined and evaluated according to a usual method by administering the composition to known animals as disease models.
For example, evaluation of the therapeutic effect on kidney fibrosis, which is a tissue fibrosis as well as a kidney disease, can be performed by a method using a renal failure model mouse (unilateral ureteral obstruction (UUO) model), in which unilateral ureteral ligation obstructs renal blood filtration in the kidney and results in renal failure in the mouse. After administration of the inventive pharmaceutical composition to the mouse, the examination is achieved by measuring the degree of inhibition of an increase of hydroxyproline production, which is an index of the onset of nephritis and kidney fibrosis induced by the renal failure. A decrease in the hydroxyproline concentration indicates the efficacy of the pharmaceutical composition for the treatment of the kidney disease.
By using the model animals described in detail in a previous report (xe2x80x9cPreparation of animals as disease models: Testing and experimental methods for the development of new drugsxe2x80x9d p. 34-46, 1993, Technological Information Society), the evaluation can be performed for kidney diseases including, for example, minimal change glomerular disease (for example, minimal change nephrotic syndrome (MCNS)), focal glomerular sclerosis (FGS), membraneous glomerulonephritis (membranous nephropathy (MN)), IgA nephropathy, mesangial proliferative glomerulonephritis, acute post-streptococcal glomerulonephritis(APSGN, crescentic (extracapillary) glomerulonephritis, interstitial nephritis, or acute renal failure.
By using the model animals described in detail in the previous report (xe2x80x9cPreparation of animals as disease models: Testing and experimental methods for the development of new drugsxe2x80x9d p. 229-235, 1993, Technological Information Society ), the evaluation can be performed for skin diseases including, for example, injuries, keloid, atopy, dermatitis, scleroderma or psoriasis.
By using the model animals described in detail in the previous report (xe2x80x9cPreparation of animals as disease models: Testing and experimental methods for the development of new drugsxe2x80x9d p. 349-358, 1993, Technological Information Society ), the evaluation can be performed for liver diseases including, for example, hepatitis (for example, viral hepatitis (type A, type B, type C, type E, etc.)), cirrhosis or drug induced hepatic injuries.
For example, the effect on arteriosclerosis and restenosis can be evaluated by using a restenosis model rat, in which the pseudo-restenosis is causeed by percutaneous transluminal coronary angioplasty(PTCA) with balloon catheter inserted in the aorta.
For example, the effect on tumor growth and metastasis can be confirmed by using mice as cancer metastasis models. The model mice are prepared by transplanting cancer cells into the subcutaneous tissue, caudal vein, spleen, tissues under the renicapsule, peritoneal cavity or cecum wall tissue, of commercially available mice including normal mice such as Balb/c mouse, or model mice such as nude mouse and SCID mouse.
The xe2x80x9crat CTGFxe2x80x9d of the present invention (specifically, having an amino acid sequence of, or substantially equivalent to that of SEQ ID NO: 2) and the xe2x80x9cDNA encoding rat CTGFxe2x80x9d (specifically, comprising the nucleotide sequence spanning from nucleotide position 213 to 1256 of the nucleotide sequence of SEQ ID NO: 1) are defined below, and can be prepared according to a usual method as shown below.
Here, the terminology xe2x80x9csubstantially equivalentxe2x80x9d has the meaning defined above.
The xe2x80x9crat CTGFxe2x80x9d of the present invention can be produced by suitably using a method known in this technical field, such as chemical synthesis and cell culture as well as the recombinant technique described below, or by using a modified method thereof.
The xe2x80x9cDNAxe2x80x9d of the present invention indicates the DNA encoding rat CTGF, and includes any nucleotide sequences as long as the nucleotide sequence encodes rat CTGF of the present invention. Specifically, the DNA includes any DNAs encoding the polypeptide with the amino acid sequence of SEQ ID NO:2. In a preferred embodiment, the DNA comprises the nucleotide sequence spanning from nucleotide position 213 to 1256 in the nucleotide sequence of SEQ ID NO: 1 (for example, the DNA having the nucleotide sequence of SEQ ID NO: 1).
The DNA of the present invention includes both cDNA and genomic DNA encoding rat CTGF.
The DNA of the present invention also includes the DNAs consisting of any codons as long as the codons encodeidentical amino acids.
The DNA of the present invention can be a DNA obtained by any method. For example, the DNA includes complementary DNA (cDNA) prepared from mRNA, DNA prepared from genomic DNA, DNA prepared by chemical synthesis, DNA obtained by PCR amplification with RNA or DNA as a template, and DNA constructed by appropriately combining these methods.
The DNA encoding the rat CTGF of the present invention can be prepared by the usual methods: cloning cDNA from MRNA encoding rat CTGF, isolating genomic DNA and splicing it, PCR using the cDNA or mRNA sequence as a template, chemical synthesis, and so on.
The DNA encoding the rat CTGF can be prepared by cleaving (digesting) each DNA encoding the rat CTGF as prepared above with an appropriate restriction enzyme, and linking the obtained DNA fragments, in combination with linker DNA or Tag if necessary, using an appropriate DNA polymerase and such.
cDNA encoding rat CTGF (hereinafter referred to as the desired protein) can be cloned from mRNA by, for example, the method described below.
First, the mRNA encoding the desired protein is prepared from tissues or cells (for example, rat fibroblasts, etc.) expressing and producing the desired protein. mRNA can be prepared by isolating total RNA by a known method such as quanidine-thiocyanate method (Chirgwin et al., Biochemistry, Vol.18, p5294, 1979), hot phenol method, or AGPC method, and subjecting it to affinity chromatography using oligo-dT cellulose or poly-U Sepharose.
Then, with the MRNA obtained as a template, cDNA is synthesized, for example, by a well-known method using reverse transcriptase, such as the method of Okayama et al (Mol. Cell. Biol. Vol.2, p.161 (1982); ibid. Vol.3, p.280 (1983)) or the method of Hoffman et al. (Gene Vol.25, p.263 (1983)), and converted into double-stranded cDNA. A cDNA library is prepared by transforming E. coli with plasmid vectors, phage vectors, or cosmid vectors having this cDNA or by transfecting E. coli after in vitro packaging.
The plasmid vectors used in this invention are not limited as long as they are replicated and maintained in hosts. Any phage vector that can be replicated in hosts can also be used. Examples of usually used cloning vectors are pUC19, xcexgt10, xcexgt11, and so on. When the vector is applied to immunological screening as mentioned below, a vector having a promoter that can express a gene encoding the desired protein in a host is preferably used.
cDNA can be inserted into a plasmid by, for example, the method of Maniatis et al. (Molecular Cloning, A Laboratory Manual, second edition, Cold Spring Harbor Laboratory, p.1.53, 1989). cDNA can be inserted into a phage vector by, for example, the method of Hyunh et al. (DNA cloning, a practical approach, Vol.1, p.49 (1985)). These methods can be simply performed by using a commercially available cloning kit (for example, a product from Takara Shuzo). The recombinant plasmid or phage vector thus obtained is introduced into an appropriate host cell such as a prokaryote (for example, E. coli: HB101, DH5xcex1, Y1090, DH10B, MC1061/P3, etc).
Examples of a method for introducing a plasmid into a host are, calcium chloride method, calcium chloride/rubidium chloride method and electroporation method, described in Molecular Cloning, A Laboratory Manual (second edition, Cold Spring Harbor Laboratory, p.1.74 (1989)). Phage vectors can be introduced into host cells by, for example, a method in which the phage DNAs are introduced into grown hosts after in vitro packaging. In vitro packaging can be easily performed with a commercially available in vitro packaging kit (for example, a product from Stratagene or Amersham).
The cDNA encoding the desired protein can be isolated from the cDNA library so prepared according to the method mentioned above by combining general cDNA screening methods.
For example, a clone comprising the desired cDNA can be screened by a known colony hybridization method (Crunstein et al. Proc. Natl. Acad. Sci. USA, Vol.72, p.3961 (1975)) or plaque hybridization method (Molecular Cloning, A Laboratory Manual, second edition, Cold Spring Harbor Laboratory, p.2.108 (1989)) using 32P-labeled chemically synthesized oligonucleotides as probes, which correspond to the amino acid sequence of the desired protein. Alternatively, a clone having a DNA fragment encoding a specific region within the desired protein can be screened by amplifying the region by PCR with synthetic PCR primers.
When a cDNA library prepared using a cDNA expression vector (for example, kgt11 phage vector) is used, the desired clone can be screened by the antigen-antibody reaction using an antibody against the desired protein. A screening method using PCR method is preferably used when many clones are subjected to screening.
The nucleotide sequence of the DNA thus obtained can be determined by Maxam-Gilbert method (Maxam et al. Proc. Natl. Acad. Sci. USA, Vol.74, p.560 (1977)) or the dideoxynucleotide synthetic chain termination method using phage M13 (Sanger et al. Proc. Natl. Acad. Sci. USA, Vol.74, pp.5463-5467 (1977)). The whole or a part of the gene encoding the desired protein can be obtained by excising the clone obtained as mentioned above with restriction enzymes and so on.
Also, the DNA encoding the desired protein can be isolated from the genomic DNA derived from the cells expressing the desired protein as mentioned above by the following methods.
Such cells are solubilized preferably by SDS or proteinase K, and the DNAs are deproteinized by repeating phenol extraction. DNAs are digested preferably with ribonuclease. The DNAs obtained are partially digested with appropriate restriction enzymes, and the DNA fragments obtained are amplified with appropriate phage or cosmid to generate a library. Then, clones having the desired sequence are detected, for example, by using radioactively labeled DNA probes, and the whole or a portion of the gene encoding the desired protein is obtained from the clones by excision with restriction enzymes etc.
A DNA encoding a desired protein can be prepared by following standard methods using known mRNA or cDNA of the desired protein as a template by means of PCR (Gene Amplification PCR method, Basics and Novel Development, Kyoritsu Publishers, 1992, etc).
A DNA encoding a desired protein can also be produced by chemical synthesis according to a usual method based on the nucleotide sequence encoding the protein.
The rat CTGF of the present invention can be prepared as a recombinant protein according to the frequently used recombinant technology by using DNA obtained by digesting the rat CTGF-encoding DNA (the cDNA or the genomic DNA comprising introns) prepared by the method indicated above with appropriate restriction enzymes; linking the resulting DNA fragment encoding the rat CTGF, according to need, with a linker DNA or Tag by using an appropriate DNA polymerase or other enzymes.
Specifically, the preparation of the protein is illustrated as follows: the DNA construct as prepared above is inserted into a vector, described below in detail, to obtain an expression vector; a host cell, which will be described hereinafter, is transformed with the expression vector to obtain a transformant; the resulting transformant cells are cultured for the production and accumulation of the desired protein in the culture supernatant; the protein accumulated in the culture supernatant can be purified easily by using column chromatography, etc.
The present invention also relates to an expression vector comprising the DNA encoding the rat CTGF of the present invention. As an expression vector of the present invention, any vector can be used as long as it is capable of retaining replication or self-multiplication in each host cell of prokaryotic and/or eukaryotic cells, including plasmid vectors and phage vectors (Cloning Vectors: A laboratory Manual, Elsevier, N.Y., 1985).
The recombinant vector can easily be prepared by ligating the DNA encoding rat CTGF of the present invention with a vector for recombination available in the art (plasmid DNA and bacteriophage DNA) by the usual method. Specific examples of the vectors for recombination used are E. coli-derived plasmids such as pBR322, pBR325, pUC12, pUC13, and pUC19, yeast-derived plasmids such as pSH19 and pSH15, and Bacillus subtilis-derived plasmids such as pUB110, pTP5, and pC194. Examples of phages are a bacteriophages such as xcex phage, and an animal or insect virus (pVL1393, Invitrogen) such as a retrovirus, vaccinia virus, and nuclear polyhedrosis virus.
A plasmid vector is useful for expressing the DNA encoding rat CTGF and for producing rat CTGF. The plasmid vector is not limited as long as it expresses the gene encoding the rat CTGF in various prokaryotic and/or eukaryotic host cells and produces this polypeptide. Examples thereof are pMAL C2, pcDNA3.1(xe2x88x92), pEF-BOS (Nucleic Acids Res. Vol.18, p.5322 (1990) and so on), pME18S (Experimental Medicine: SUPPLEMENT, xe2x80x9cHandbook of Genetic Engineeringxe2x80x9d (1992) and so on), etc.
When bacteria, particularly E. coli are used as host cells, an expression vector is generally comprised of, at least, a promoter/operator region, an initiation codon, the DNA encoding the protein of the present invention, termination codon, terminator region, and replicon.
When yeast, animal cells, or insect cells are used as hosts, an expression vector is preferably comprised of, at least, a promoter, an initiation codon, the DNA encoding the rat CTGF of the present invention, and a termination codon. It may also comprise the DNA encoding a signal peptide, enhancer sequence, 5xe2x80x2- and 3 xe2x80x2-untranslated region of the gene encoding the rat CTGF of the present invention, splicing junctions, polyadenylation site, selectable marker region, and replicon. The expression vector may also contain, if required, a gene for gene amplification (marker) that is usually used.
A promoter/operator region to express the fusion polypeptide of the present invention in bacteria comprises a promoter, an operator, and a Shine-Dalgarno (SD) sequence (for example, AAGG). For example, when the host is Escherichia, it preferably comprises Trp promoter, lac promoter, recA promoter, xcexPL promoter, 1pp promoter, tac promoter, or the like.
Examples of a promoter to express the rat CTGF of the present invention in yeast are PH05 promoter, PGK promoter, GAP promoter, ADH promoter, and so on. When the host is Bacillus, examples thereof are SL01 promoter, SP02 promoter, penP promoter and so on.
When the host is a eukaryotic cell such as a mammalian cell, examples thereof are SV40-derived promoter, retrovirus promoter, heat shock promoter, and soon, and preferably SV-40 and retrovirus-derived one. As a matter of course, the promoter is not limited to the above examples. In addition, using an enhancer is effective for expression.
A preferable initiation codon is, for example, a methionine codon (ATG).
A commonly used termination codon (for example, TAG, TAA, TGA) is exemplified as a termination codon.
Usually, used natural or synthetic terminators are used as a terminator region.
A replicon means a DNA capable of replicating the whole DNA sequence in host cells, and includes a natural plasmid, an artificially modified plasmid (DNA fragment prepared from a natural plasmid), a synthetic plasmid, and so on. Examples of preferable plasmids are pBR322 or its artificial derivatives (DNA fragment obtained by treating pBR322 with appropriate restriction enzymes) for E. coli, yeast 2xcexc plasmid or yeast chromosomal DNA for yeast, and pRSVneo ATCC 37198, pSV2dhfr ATCC 37145, pdBPV-MMTneo ATCC 37224, pSV2neo ATCC 37149, pSV2bsr, and such for mammalian cells.
An enhancer sequence, polyadenylation site, and splicing junction that are usually used in the art, such as those derived from SV40 can also be used.
A selectable marker usually employed can be used according to the usual method. Examples thereof are resistance genes for antibiotics, such as tetracycline, ampicillin, or kanamycin.
Examples of genes for gene amplification are dihydrofolate reductase (DHFR) gene, thymidine kinase gene, neomycin resistance gene, glutamate synthase gene, adenosine deaminase gene, ornithine decarboxylase gene, hygromycin-B-phophotransferase gene, aspartate transcarbamylase gene, etc.
The expression vector of the present invention can be prepared by continuously and circularly linking at least the above-mentioned promoter, initiation codon, DNA encoding the protein of the present invention, termination codon, and terminator region, to an appropriate replicon. If desired, appropriate DNA fragments (for example, linkers, restriction sites generated with other restriction enzyme), can be used by the usual method such as digestion with a restriction enzyme or ligation using T4 DNA ligase.
Transformants of the present invention can be prepared by introducing the expression vector mentioned above into host cells.
Host cells used in the present invention are not limited as long as they are compatible with an expression vector mentioned above and can be transformed. Examples thereof are various cells such as wild-type cells or artificially established recombinant cells usually used in technical field of the present invention (for example, bacteria (Escherichia and Bacillus), yeast (Saccharomyces, Pichia, and such), animal cells, or insect cells).
E. coli or animal cells are preferably used. Specific examples are E. coli (DH5 xcex1, DH10B, TB1, HB101, XL-2Blue, and such), mouse-derived cells (COP, L, C127, Sp2/0, NS-1, NIH 3T3, and such), rat-derived cells, hamster-derived cells (BHK, CHO, and such), monkey-derived cells (COS1, COS3, COS7, CV1, Velo, and such), and human-derived cells (Hela, diploid fibroblast-derived cells, myeloma, Namalwa, and such).
An expression vector can be introduced (transformed (transduced)) into host cells by known methods.
Transformation can be performed, for example, according to the method of Cohen et al. (Proc. Natl. Acad. Sci. USA, Vol.69, p.2110 (1972)), protoplast method (Mol. Gen. Genet., Vol.168, p.111 (1979)), or competent method (J. Mol. Biol., Vol.56, p.209 (1971)) when the hosts are bacteria (E. coli, Bacillus subtilis, and such), the method of Hinnen et al. (Proc. Natl. Acad. Sci. USA, Vol.75, p.1927 (1978)), or lithium method (J. Bacteriol., Vol.153, p.163 (1983)) when the host is Saccharomyces cerevisiae, the method of Graham (Virology, Vol.52, p.456 (1973)) when the hosts are animal cells, and the method of Summers et al. (Mol. Cell. Biol., Vol.3, pp.2156-2165 (1983)) when the hosts are insect cells.
Rat CTGF of the present invention can be produced by cultivating transformants (in the following this term includes transductants) comprising an expression vector prepared as mentioned above in nutrient media.
The nutrient media preferably comprise carbon source, inorganic nitrogen source, or organic nitrogen source necessary for the growth of host cells (transformants). Examples of the carbon source are glucose, dextran, soluble starch, and sucrose, and examples of the inorganic or organic nitrogen source are ammonium salts, nitrates, amino acids, corn steep liquor, peptone, casein, meet extract, soy bean cake, and potato extract. If desired, they may comprise other nutrients (for example, an inorganic salt (for example, calcium chloride, sodium dihydrogenphosphate, and magnesium chloride), vitamins, antibiotics (for example, tetracycline, neomycin, ampicillin, kanamycin, and so on).
Cultivation is performed by a method known in the art. Cultivation conditions such as temperature, pH of the media, and cultivation time are selected appropriately so that the protein of the present invention is produced in large quantities.
Specific media and cultivation conditions used depending on host cells are illustrated below, but are not limited thereto.
When the hosts are bacteria, actinomycetes, yeasts, filamentous fungi, liquid media comprising the nutrient source mentioned above are appropriate. The media with pH 5 to 8 are preferably used. When the host is E. coli, examples of preferable media are LB media, M9media (Milleretal. Exp. Mol. Genet., Cold Spring Harbor Laboratory, p.431 (1972)), YT medium, and so on. Using these media, cultivation can be performed usually at 14 to 43xc2x0 C. for about 3 to 24 hours with aeration and stirring, if necessary.
When the host is Bacillus, cultivation can be performed usually at 30 to 40xc2x0 C. for about 16 to 96 hours with aeration and stirring, if necessary.
When the host is yeast, an example of media is Burkholder minimal media (Bostian, Proc. Natl. Acad. Sci. USA, Vol.77, p.4505 (1980)). The pH of the media is preferably 5 to 8. Cultivation can be performed usually at 20 to 35xc2x0 C. for about 14 to 144 hours with aeration and stirring, if necessary.
When the host is an animal cell, examples of media are MEM media containing about 5 to 20% fetal bovine serum (Science, Vol.122, p.501 (1952)), DMEM media (Virology, Vol.8, p.396 (1959)), RPMI1640 media (J. Am. Med. Assoc., Vol.199, p.519 (1967)), 199 media (Proc. Soc. Exp. Biol. Med., Vol.73, p.1 (1950)), HamF12 media, and so on. The pH of the media is preferably about 6 to 8. Cultivation can be performed usually at about 30 to 40xc2x0 C. for about 15 to 72 hours with aeration and stirring, if necessary.
When the host is an insect cell, an example of media is Grace""s media containing fetal bovine serum (Proc. Natl. Acad. Sci. USA, Vol.82, p.8404 (1985)). The pH thereof is preferably about 5 to 8. Cultivation can be performed usually at about 20 to 40xc2x0 C. for 15 to 100 hours with aeration and stirring, if necessary.
Rat CTGF of the present invention can be produced by cultivating transformants as mentioned above (in particular animal cells or E. coli) and allowing them to secrete the protein into the culture supernatant. Namely, a culture filtrate (supernatant) is obtained by a method such as filtration or centrifugation of the obtained culture, and the rat CTGF of the present invention is purified and isolated from the culture filtrate by methods commonly used in order to purify and isolate a natural or synthetic protein.
Examples of the isolation and purification method are a method utilizing affinity, such as affinity column chromatography; a method utilizing solubility, such as salting out and solvent precipitation method; a method utilizing the difference in molecular weight, such as dialysis, ultrafiltration, gel filtration, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis; a method utilizing charges, such as ion exchange chromatography and hydroxylapatite chromatography; a method utilizing the difference in hydrophobicity, such as reverse phase high performance liquid chromatography; and a method utilizing the difference in isoelectric point, such as isoelectric focusing.
When the rat CTGF of the present invention exists in the periplasm or cytoplasm of cultured transformants, first, the cells are harvested by a usual method such as filtration or centrifugation and suspended in appropriate buffer. After the cell wall and/or cell membrane of the cells and such are disrupted by the method such as lysis with sonication, lysozyme, and freeze-thawing, the membrane fraction comprising the rat CTGF of the present invention is obtained by the method such as centrifugation or filtration. The membrane fraction is solubilized with a detergent such as Triton-X100 to obtain the crude extract. Finally, the protein is isolated and purified from the crude extract by the usual method as illustrated above.
The xe2x80x9ctransgenic mousexe2x80x9d of the present is a transgenic mouse in which the above human CTGF encoding DNA (cDNA or genomic DNA) prepared by the method mentioned above has been integrated into the endogenous gene locus of the mouse. This transgenic mouse expresses and secretes the human CTGF in vivo.
The transgenic mouse can be prepared according to the method usually used for producing a transgenic animal (for example, see xe2x80x9cNewest Manual of Animal Cell Experimentxe2x80x9d, LIC press, Chapter 7, pp.361-408, (1990)). Specifically, for example, a transgenic mouse can be produced as follows. Embryonic stem cells (ES cells) obtained from normal mouse blastocysts are transformed with an expression vector in which the gene encoding the human CTGF has been inserted in an expressible manner. ES cells in which the gene encoding the human CTGF has been integrated into the endogenous gene are screened by a usual method. Then, the ES cells screened are microinjected into a fertilized egg (blastocyst) obtained from another normal mouse (Proc. Natl. Acad. Sci. USA, Vol.77, No.12, pp.7380-7384 (1980); U.S. Pat. No. 4,873,191). The blastocyst is transplanted into the uterus of another normal mouse as the foster mother and chimeric transgenic mice are born. By mating the chimeric transgenic mice with normal mice, heterozygous transgenic mice are obtained. By mating the heterozygous transgenic mice with each other, homozygous transgenic mice are obtained according to Mendel""s laws.